Bioreactor Expansion of Human Mesenchymal Stem Cells According to GMP Requirements

Elseberg, Christiane; Salzig, Denise; Czermak, Peter

doi:10.1007/7651_2014_117

Cited by 14 publications

(9 citation statements)

References 14 publications

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“…After differentiation, the cells were fixed with 4% paraformaldehyde (Carl Roth) for 30 min at room temperature. Adipogenic differentiation was confirmed by nil red staining of the fat droplets as previously described [ 25 ]. Finally, the sample was embedded with Mowiol (Carl Roth) according to the manufacturer's recommendations.…”

Section: Methodsmentioning

confidence: 99%

Attachment, Growth, and Detachment of Human Mesenchymal Stem Cells in a Chemically Defined Medium

Salzig

Leber

Merkewitz

et al. 2016

Stem Cells International

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The manufacture of human mesenchymal stem cells (hMSCs) for clinical applications requires an appropriate growth surface and an optimized, preferably chemically defined medium (CDM) for expansion. We investigated a new protein/peptide-free CDM that supports the adhesion, growth, and detachment of an immortalized hMSC line (hMSC-TERT) as well as primary cells derived from bone marrow (bm-hMSCs) and adipose tissue (ad-hMSCs). We observed the rapid attachment and spreading of hMSC-TERT cells and ad-hMSCs in CDM concomitant with the expression of integrin and actin fibers. Cell spreading was promoted by coating the growth surface with collagen type IV and fibronectin. The growth of hMSC-TERT cells was similar in CDM and serum-containing medium whereas the lag phase of bm-hMSCs was prolonged in CDM. FGF-2 or surface coating with collagen type IV promoted the growth of bm-hMSCs, but laminin had no effect. All three cell types retained their trilineage differentiation capability in CDM and were detached by several enzymes (but not collagenase in the case of hMSC-TERT cells). The medium and coating did not affect detachment efficiency but influenced cell survival after detachment. CDM combined with cell-specific surface coatings and/or FGF-2 supplements is therefore as effective as serum-containing medium for the manufacture of different hMSC types.

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Section: Methodsmentioning

confidence: 99%

Attachment, Growth, and Detachment of Human Mesenchymal Stem Cells in a Chemically Defined Medium

Salzig

Leber

Merkewitz

et al. 2016

Stem Cells International

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“…Current technologies for pluripotent stem cell expansion hardly fulfil the demands of regulatory boards (Kurtz et al, 2014) and the needs of clinical use (Kamao et al, 2014), as they are associated with low cost-efficiency (Jenkins & Farid, 2015), lack stringent quality control, or do not meet good manufacturing practices (GMP) standards (Elseberg, Salzig, & Czermak, 2015). Therefore, the development of a standardized, scalable method for pluripotent stem cell expansion that is cost-effective and compatible with automation is of great interest.…”

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confidence: 99%

Scalable stirred suspension culture for the generation of billions of human induced pluripotent stem cells using single‐use bioreactors

Kwok

Ueda

Kadari

et al. 2017

J Tissue Eng Regen Med

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The production of human induced pluripotent stem cells (hiPSCs) in quantities that are relevant for cell-based therapies and cell-loaded implants through standard adherent culture is hardly achievable and lacks process scalability. A promising approach to overcoming these hurdles is the culture of hiPSCs in suspension. In this study, stirred suspension culture vessels were investigated for their suitability in the expansion of two hiPSC lines inoculated as a single cell suspension, with a free scalability between volumes of 50 and 2400 ml. The simple and robust two-step process reported here first generates hiPSC aggregates of 324 ± 71 μm diameter in 7 days in 125 ml spinner flasks (100 ml volume). These are subsequently dissociated into a single cell suspension for inoculation in 3000 ml bioreactors (1000 ml volume), finally yielding hiPSC aggregates of 198 ± 58 μm after 7 additional days. In both spinner flasks and bioreactors, hiPSCs can be cultured as aggregates for more than 40 days in suspension, maintain an undifferentiated state as confirmed by the expression of pluripotency markers TRA-1-60, TRA-1-81, SSEA-4, OCT4, and SOX2, can differentiate into cells of all three germ layers, and can be directed to differentiate into specific lineages such as cardiomyocytes. Up to a 16-fold increase in hiPSC quantity at the 100 ml volume was achieved, corresponding to a fold increase per day of 2.28; at the 1000 ml scale, an additional 10-fold increase was achieved. Taken together, 16 × 10 6 hiPSCs were expanded into 2 × 10 9 hiPSCs in 14 days for a fold increase per day of 8.93. This quantity of hiPSCs readily meets the requirements of cell-based therapies and brings their clinical potential closer to fruition.

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“…Having said that, what is the remedy for using cells having a low starting material such as dental pulp or periodontal ligament tissue? To overcome this issue, large expansions of cells using a 3D bioreactor [31] are possibly made to dental stem cells at the early passage without triggering any side effects [32]. Apart from that, pooled allogeneic samples are another choice, but further characterization needs to be done to maintain their functionality.…”

Section: Discussionmentioning

confidence: 99%

Expression patterns of immune genes in long-term cultured dental stem cells

et al. 2015

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Bioreactor Expansion of Human Mesenchymal Stem Cells According to GMP Requirements

Cited by 14 publications

References 14 publications

Attachment, Growth, and Detachment of Human Mesenchymal Stem Cells in a Chemically Defined Medium

Attachment, Growth, and Detachment of Human Mesenchymal Stem Cells in a Chemically Defined Medium

Scalable stirred suspension culture for the generation of billions of human induced pluripotent stem cells using single‐use bioreactors

Expression patterns of immune genes in long-term cultured dental stem cells

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