Microcarrier-based Expansion Process for hMSCs with High Vitality and Undifferentiated Characteristics

Elseberg, Christiane; Leber, Jasmin; Salzig, Denise; Wallrapp, Christine; Kassem, Moustapha; Kraume, Matthias; Czermak, Peter

doi:10.5301/ijao.5000077

Cited by 44 publications

(44 citation statements)

References 35 publications

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“…At the end of the cultivation, a cell density of around 50 000 cells per cm 2 growth surface, a total cells number up to 4 · 10 8 cells per L of production volume, an expansion factor of 7.2 and a cell yield after harvest of over 95 % was determined. Quality control of the harvested hMSC-TERT showed high viabilities and differentiability [60].…”

Section: Expansion Of Hmscs In Dynamic Systemsnew Conceptsmentioning

confidence: 99%

hMSC Production in Disposable Bioreactors with Regards to GMP and PAT

Cierpka

Elseberg

Niss³

et al. 2012

Chemie Ingenieur Technik

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Reactor concepts for human mesenchymal stem cell (hMSC) production are introduced. Thereby, special interest is laid on the realization of these concepts as disposables fulfilling the GMP and PAT requirements. The specialty of the hMSC production process is the cell itself being the product. This results in completely different process requirements compared to e.g. protein production in mammalian cells. Thus, great attention has to be given to the shear sensitivity of the cells. The cultivation and the harvest of the cells have to be very gentle to neither influence cell viability nor cell differentiability. Further, the production process should not cause any undesirable cell changes. For hMSC production, cell harvest is the main challenging process step. The reactor concepts should be suitable for hMSC production for clinical trials as ATMPs. Therefore, disposable systems are especially applicable. The review describes more detailed bone marrow-derived hMSC production in a disposable stirred tank reactor as promising reactor concept.

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Section: Expansion Of Hmscs In Dynamic Systemsnew Conceptsmentioning

confidence: 99%

hMSC Production in Disposable Bioreactors with Regards to GMP and PAT

Cierpka

Elseberg

Niss³

et al. 2012

Chemie Ingenieur Technik

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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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“…Microcarriers 2-5 mm in diameter are often used for FBR processes, whereas those used in fluidized bed reactors are typically 1 mm in diameter and those used in STRs are generally 100-300 µm in diameter [80]. Glass has been used as a cell culture growth surface for decades [81], and low-density microcarriers with a copolymer plastic core and a high-silica glass coating were used for the expansion of hMSC-TERT in serum-containing medium [51,82]. These results are based on a microcarrier choice for the hMSC-TERT cell line resulting in strong proliferation and a good yield of detached cells for glass and polystyrene microcarriers [36].…”

Section: Microcarrier Choicementioning

confidence: 99%

“…Initial seeding densities of 1-3 × 10 4 cells/cm 2 are nearly independent from the available growth surface area. The inoculation strategy is often based on intermittent agitation or flow depending on the system [50,51].…”

Section: Cell Expansion In Bioreactorsmentioning

confidence: 99%

“…Intensive work on the development of a hMSC-TERT batch process in a 3 L glass STR with a working volume of 1.65 L was described by Elseberg and colleagues, and the cell characteristics were retained [39,51]. The sterilized bioreactor (Figure 4) was filled with the sterile glass-surface microcarrier RapidCell [36] at 25 g/L suspended in high-glucose DMEM supplemented with 10% serum [51].…”

Section: Stirred Tank Reactormentioning

confidence: 99%

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The Challenge of Human Mesenchymal Stromal Cell Expansion: Current and Prospective Answers

Elseberg¹,

Leber²,

Weidner³

et al. 2017

New Insights Into Cell Culture Technology

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In the field of cell therapy, allogenic human mesenchymal stromal cells (hMSCs) are often used in clinical trials, creating a demand for cell mass production using efficient dynamic bioreactor systems. As an advanced therapy medicinal product (ATMP), such cells should meet certain special requirements, including product specifications requiring a production process compatible with good manufacturing practice (GMP). The development of processes in which the cells are the product therefore remains a significant challenge. This chapter describes the requirements at different steps in the upstream and downstream phases of such dynamic processes. Potential solutions are presented and future prospects are discussed, including the selection of media and carriers for the strictly adherent growing cells, allowing efficient cell adhesion and detachment. Strategies for dynamic cultivation in bioreactors are described in detail for fixed-bed and stirred-tank reactors based on GMP requirements and the integration of process analytical technology (PAT). Following cell harvest, separation and purification, the formulation and storage of the product are also described. Finally, the chapter covers important cell quality characteristics necessary for the approval of ATMPs.

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Microcarrier-based Expansion Process for hMSCs with High Vitality and Undifferentiated Characteristics

Cited by 44 publications

References 35 publications

hMSC Production in Disposable Bioreactors with Regards to GMP and PAT

hMSC Production in Disposable Bioreactors with Regards to GMP and PAT

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

The Challenge of Human Mesenchymal Stromal Cell Expansion: Current and Prospective Answers

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