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GMP Production and Scale-Up of Adherent Neural Stem Cells with a Quantum Cell Expansion System.

ABSTRACT: Cell-based therapies hold great promise for a myriad of clinical applications. However, as these therapies move from phase I to phase II and III trials, there is a need to improve scale-up of adherent cells for the production of larger good manufacturing practice (GMP) cell banks. As we advanced our neural stem cell (NSC)-mediated gene therapy trials for glioma to include dose escalation and multiple treatment cycles, GMP production using cell factories (CellStacks) generated insufficient neural stem cell (NSC) yields. To increase yield, we developed an expansion method using the hollow fiber quantum cell expansion (QCE) system. Seeding of 5.2 × 107 NSCs in a single unit yielded up to 3 × 109 cells within 10 days. These QCE NSCs showed genetic and functional stability equivalent to those expanded by conventional flask-based methods. We then expanded the NSCs in 7 units simultaneously to generate a pooled GMP-grade NSC clinical lot of more than 1.5 × 1010 cells in only 9 days versus 8 × 109 over 6 weeks in CellStacks. We also adenovirally transduced our NSCs within the QCE. We found the QCE system enabled rapid cell expansion and increased yield while maintaining cell properties and reducing process time, labor, and costs with improved efficiency and reproducibility.

SUBMITTER: Tirughana R

PROVIDER: S-EPMC6037686 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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GMP Production and Scale-Up of Adherent Neural Stem Cells with a Quantum Cell Expansion System.

Tirughana Revathiswari R Metz Marianne Z MZ Li Zhongqi Z Hall Christine C Hsu David D Beltzer Jim J Annala Alexander J AJ Oganesyan Diana D Gutova Margarita M Aboody Karen S KS

Molecular therapy. Methods & clinical development 20180707

Cell-based therapies hold great promise for a myriad of clinical applications. However, as these therapies move from phase I to phase II and III trials, there is a need to improve scale-up of adherent cells for the production of larger good manufacturing practice (GMP) cell banks. As we advanced our neural stem cell (NSC)-mediated gene therapy trials for glioma to include dose escalation and multiple treatment cycles, GMP production using cell factories (CellStacks) generated insufficient neural ...[more]

PMID: 29992178

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Project description:BackgroundMesenchymal stromal cells (MSC) have gained importance in tissue repair, tissue engineering and in immunosupressive therapy during the last years. Due to the limited availability of MSC in the bone marrow, ex vivo amplification prior to clinical application is requisite to obtain therapeutic applicable cell doses. Translation of preclinical into clinical-grade large-scale MSC expansion necessitates precise definition and standardization of all procedural parameters including cell seeding density, culture medium and cultivation devices. While xenogeneic additives such as fetal calf serum are still widely used for cell culture, its use in the clinical context is associated with many risks, such as prion and viral transmission or adverse immunological reactions against xenogeneic components.Methods and findingsWe established animal-free expansion protocols using platelet lysate as medium supplement and thereby could confirm its safety and feasibility for large-scale MSC isolation and expansion. Five different GMP-compliant standardized protocols designed for the safe, reliable, efficient and economical isolation and expansion of MSC was performed and MSC obtained were analyzed for differentiation capacity by qPCR and histochemistry. Expression of standard MSC markers as defined by the International Society for Cellular Therapy as well as expression of additional MSC markers and of various chemokine and cytokine receptors was analysed by flow cytometry. Changes of metabolic markers and cytokines in the medium were addressed using the LUMINEX platform.ConclusionsThe five different systems for isolation and expansion of MSC described in this study are all suitable to produce at least 100 millions of MSC, which is commonly regarded as a single clinical dose. Final products are equal according to the minimal criteria for MSC defined by the ISCT. We showed that chemokine and integrin receptors analyzed had the same expression pattern, suggesting that MSC from either of the systems show equal characteristics of homing and adhesion.

Project description:BackgroundHuman embryonic stem cells (hESCs) offer a virtually unlimited source of neural cells for structural repair in neurological disorders, such as stroke. Neural cells can be derived from hESCs either by direct enrichment, or by isolating specific growth factor-responsive and expandable populations of human neural stem cells (hNSCs). Studies have indicated that the direct enrichment method generates a heterogeneous population of cells that may contain residual undifferentiated stem cells that could lead to tumor formation in vivo.Methods/principal findingsWe isolated an expandable and homogenous population of hNSCs (named SD56) from hESCs using a defined media supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and leukemia inhibitory growth factor (LIF). These hNSCs grew as an adherent monolayer culture. They were fully neuralized and uniformly expressed molecular features of NSCs, including nestin, vimentin and radial glial markers. These hNSCs did not express the pluripotency markers Oct4 or Nanog, nor did they express markers for the mesoderm or endoderm lineages. The self-renewal property of the hNSCs was characterized by a predominant symmetrical mode of cell division. The SD56 hNSCs differentiated into neurons, astrocytes and oligodendrocytes throughout multiple passages in vitro, as well as after transplantation. Together, these criteria confirm the definitive NSC identity of the SD56 cell line. Importantly, they exhibited no chromosome abnormalities and did not form tumors after implantation into rat ischemic brains and into naïve nude rat brains and flanks. Furthermore, hNSCs isolated under these conditions migrated toward the ischemia-injured adult brain parenchyma and improved the independent use of the stroke-impaired forelimb two months post-transplantation.Conclusions/significanceThe SD56 human neural stem cells derived under the reported conditions are stable, do not form tumors in vivo and enable functional recovery after stroke. These properties indicate that this hNSC line may offer a renewable, homogenous source of neural cells that will be valuable for basic and translational research.

Dataset Information

GMP Production and Scale-Up of Adherent Neural Stem Cells with a Quantum Cell Expansion System.

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GMP Production and Scale-Up of Adherent Neural Stem Cells with a Quantum Cell Expansion System.

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