Production Process for Stem Cell Based Therapeutic Implants: Expansion of the Production Cell Line and Cultivation of Encapsulated Cells

Weber, Christian; Pöhl, Sebastian; Poertner, Ralf; Pino-Grace, Pablo; Freimark, Denise; Wallrapp, Christine; Geigle, Peter; Czermak, Peter

doi:10.1007/10_2009_25

Cited by 26 publications

(37 citation statements)

References 23 publications

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“…These eMSCs secrete endogenous paracrine factors that include vascular endothelial growth factor, monocyte chemotactic protein-1, interleukin (IL)-6, IL-8, glial-derived neurotrophic factor, and neurotrophin-3 and are genetically modified to produce glucagon-like peptide-1 (GLP-1) fusion protein, which comprises 2 GLP-1 molecules bound by an intervening peptide, extending its half-life in vivo. [8][9][10][11][12] Among its beneficial effectsBackground-Engraftment and survival of stem cells in the infarcted myocardium remain problematic in cell-based therapy for cardiovascular disease. To overcome these issues, encapsulated mesenchymal stem cells (eMSCs) were developed that were transfected to produce glucagon-like peptide-1, an incretin hormone with known cardioprotective effects, alongside MSC endogenous paracrine factors.…”

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confidence: 99%

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Intracoronary Infusion of Encapsulated Glucagon-Like Peptide-1–Eluting Mesenchymal Stem Cells Preserves Left Ventricular Function in a Porcine Model of Acute Myocardial Infarction

Jong

Hout

Houtgraaf

et al. 2014

Circ: Cardiovascular Interventions

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R egenerative stem cell therapy to promote cardiac repair has been a target of interest in the past 10 years to prevent heart failure after an acute myocardial infarction (AMI). 1,2 Various different stem cells have been investigated for their ability to repair the heart.3 Mesenchymal stem cells (MSCs) seem to be a potent candidate to date. The mechanism of action of MSC is primarily based on the release of paracrine factors to the myocardium. 4 However, retention and survival of stem cells in the myocardium after intracoronary infusion remains an issue in cell-based therapy, because only a limited number of surviving stem cells remain in the myocardium, thereby limiting the potential benefit of the therapy. [5][6][7] CellBeads, which consist of alginate-encapsulated MSCs (eMSCs; BTG International Germany GmbH, Alzenau, Germany), were developed to improve survival of cells in the myocardium, thereby elongating the release of cardioprotective proteins into the infarcted myocardium. These eMSCs secrete endogenous paracrine factors that include vascular endothelial growth factor, monocyte chemotactic protein-1, interleukin (IL)-6, IL-8, glial-derived neurotrophic factor, and neurotrophin-3 and are genetically modified to produce glucagon-like peptide-1 (GLP-1) fusion protein, which comprises 2 GLP-1 molecules bound by an intervening peptide, extending its half-life in vivo. [8][9][10][11][12] Among its beneficial effectsBackground-Engraftment and survival of stem cells in the infarcted myocardium remain problematic in cell-based therapy for cardiovascular disease. To overcome these issues, encapsulated mesenchymal stem cells (eMSCs) were developed that were transfected to produce glucagon-like peptide-1, an incretin hormone with known cardioprotective effects, alongside MSC endogenous paracrine factors. This study was designed to investigate the efficacy of different doses of intracoronary infusion of eMSC in a porcine model of acute myocardial infarction (AMI). Methods and Results-One hundred pigs were subjected to a moderate AMI (posterolateral AMI; n=50) or a severe AMI (anterior AMI; n=50), whereupon surviving animals (n=36 moderate, n=33 severe) were randomized to receive either intracoronary infusion of 3 incremental doses of eMSC or Ringers' lactate control. Cardiac function was assessed using invasive hemodynamics, echocardiography, and histological analysis. A trend was observed in the moderate AMI model, whereas in the severe AMI model, left ventricular ejection fraction improved by +9.3% (P=0.004) in the best responding eMSC group, because of a preservation of left ventricular end-systolic volume. Arteriolar density increased 3-fold in the infarct area (8.4±0.9/mm 2 in controls versus 22.2±2.6/mm 2 in eMSC group; P<0.001). Although not statistically significant, capillary density was 30% higher in the border zone (908.1±99.7/mm 2 in control versus 1209.0±64.6/mm 2 in eMSC group; P=ns). Conclusions-eMSCs enable sustained local delivery of cardioprotective proteins to the heart, thereby enhancing angio...

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confidence: 99%

mentioning

confidence: 99%

Intracoronary Infusion of Encapsulated Glucagon-Like Peptide-1–Eluting Mesenchymal Stem Cells Preserves Left Ventricular Function in a Porcine Model of Acute Myocardial Infarction

Jong

Hout

Houtgraaf

et al. 2014

Circ: Cardiovascular Interventions

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“…For example for hMSC-TERT ATMP production, it is not only important to efficiently detach cells, but also to keep them viable (and undifferentiated). This gets challenging when production takes places in dynamic systems (stirred tank reactors (Cierpka et al, 2013;Elseberg et al, 2012), fixed bed-systems (Weber et al, 2010a;2010b;2010c) and the harvested cells are further processed (e.g., beadto-bead transfer, cell encapsulation (Freimark et al, 2010). Trypsin is unsuitable for hMSC-TERT detachment out of dynamic systems and for detachment of further processed cells.…”

Section: Discussionmentioning

confidence: 99%

CELL DETACHMENT BY PROLYL-SPECIFIC ENDOPEPTIDASE FROM <i>WOLFIPORIA COCOS</i>

Mika

Cierpka

Lange

et al. 2014

American Journal of Biochemistry and Biotechnology

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As requirements for Advanced Therapy Medicinal Product (ATMP) production differ from other production processes (e.g., therapeutic protein production), cell detachment is often a crucial step for the process success. In most cases, cell detachment is done enzymatically. Although many peptidases are established in cell culture in R&D, e.g., Trypsin as gold standard, many of them seem to be unsuitable in ATMP production processes. Therefore, the present study investigated a novel endopeptidase used in food biotechnology for its applicability in ATMP processes where cell detachment is needed. The Prolyl-specific Peptidase (PsP) is of nonmammalian origin and considered as safe for humans. PsP was purified from the supernatant of the fungus Wolfiporia cocos. The isolation and purification resulted in an enzyme solution with 0.19 U mg −1 prolylspecific activity. By in silico analysis it was confirmed that attachment-promoting proteins can be cleaved by PsP in a similar amount than with Trypsin. Further the proteolytic activity was determined for PsP and Trypsin by using the same enzymatic assay. Detachment with both enzymes was compared for cells used in typical therapeutic production processes namely a mesenchymal stem cell line (hMSC-TERT) as a model for a cell therapeutic, Vero and MA104 cells used for viral therapeutic or vaccine production. The cell detachment experiments were performed with comparable enzyme activities (1.6 U mL −1). hMSC-TERT detachment was faster with PsP than with Trypsin. For Vero cells the detachment with PsP was not only faster but also more efficient. For MA104 cells the detachment rate with PsP was similar to Trypsin. For all cell types, detachment with PsP showed less influence on cell growth and metabolism compared to standard Trypsin.Thus, three cell types used in ATMP, viral therapeutics or vaccine production can be detached efficiently and gently with PsP. Therefore, PsP shows potential for cell detachment in ATMP and viral/vaccine production processes.

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“…Whole cells may also be the target product for example in stem cell therapy (Weber et al 2007b(Weber et al , 2010aFreimark et al 2010). Standard cell cultivation systems for cell expansion and production of biopharmaceuticals on microcarrier are described in a variety of culture systems (Eibl et al 2008).…”

Section: Introductionmentioning

confidence: 99%

Online- and offline- monitoring of stem cell expansion on microcarrier

et al. 2011

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In the biopharmaceutical industry, adherent growing stem cell cultures gain worldwide importance as cell products. The cultivation process of these cells, such as in stirred tank reactors or in fixed bed reactors, is highly sophisticated. Cultivations need to be monitored and controlled to guarantee product quality and to satisfy GMP requirements. With the process analytical technology (PAT) initiative, requirements regarding process monitoring and control have changed and real-time on-line monitoring tools are recommended. A tool meeting the new requirements may be the dielectric spectroscopy for online viable cell mass determination by measurement of the permittivity. To establish these tools, proper offline methods for data correlation are required. The cell number determination of adherent cells on microcarrier is difficult, as it requires cell detachment from the carrier, which highly increases the statistical error. As an offline method, a fluorescence assay based on SYBR Ò GreenI was developed allowing fast and easy total cell concentration determination without the need to detach the cells from the carrier. The assay is suitable for glass carriers used in stirred tank reactor systems or in fixed bed systems, may be suitable for different cell lines and can be applied to high sample numbers easily. The linear dependency of permittivity to cell concentration of suspended stem cells with the dielectric spectroscopy is shown for even very small cell concentrations. With this offline-method, a correlation of the cell concentration grown on carrier to the permittivity data measured by the dielectric spectroscopy was done successfully.

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Production Process for Stem Cell Based Therapeutic Implants: Expansion of the Production Cell Line and Cultivation of Encapsulated Cells

Cited by 26 publications

References 23 publications

Intracoronary Infusion of Encapsulated Glucagon-Like Peptide-1–Eluting Mesenchymal Stem Cells Preserves Left Ventricular Function in a Porcine Model of Acute Myocardial Infarction

Intracoronary Infusion of Encapsulated Glucagon-Like Peptide-1–Eluting Mesenchymal Stem Cells Preserves Left Ventricular Function in a Porcine Model of Acute Myocardial Infarction

CELL DETACHMENT BY PROLYL-SPECIFIC ENDOPEPTIDASE FROM <i>WOLFIPORIA COCOS</i>

Online- and offline- monitoring of stem cell expansion on microcarrier

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