Factorial Experimental Design for the Culture of Human Embryonic Stem Cells as Aggregates in Stirred Suspension Bioreactors Reveals the Potential for Interaction Effects Between Bioprocess Parameters

Hunt, Megan M.; Meng, Guoliang; Rancourt, Derrick E.; Gates, Ian D.; Kallos, Michael S.

doi:10.1089/ten.tec.2013.0040

Cited by 53 publications

(47 citation statements)

References 28 publications

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“…Abbasalizadeh et al [69] could maintain the aggregate size very homogeneously, ranging from 190 to 215 mm. This size range was also achieved by Hunt et al [121] with an agitation of 100 r.p.m.…”

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confidence: 67%

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Advances in cell culture: anchorage dependence

Merten

2015

Phil. Trans. R. Soc. B

128

103

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Anchorage-dependent cells are of great interest for various biotechnological applications. (i) They represent a formidable production means of viruses for vaccination purposes at very large scales (in 1000-6000 l reactors) using microcarriers, and in the last decade many more novel viral vaccines have been developed using this production technology. (ii) With the advent of stem cells and their use/potential use in clinics for cell therapy and regenerative medicine purposes, the development of novel culture devices and technologies for adherent cells has accelerated greatly with a view to the large-scale expansion of these cells. Presently, the really scalable systems-microcarrier/ microcarrier-clump cultures using stirred-tank reactors-for the expansion of stem cells are still in their infancy. Only laboratory scale reactors of maximally 2.5 l working volume have been evaluated because thorough knowledge and basic understanding of critical issues with respect to cell expansion while retaining pluripotency and differentiation potential, and the impact of the culture environment on stem cell fate, etc., are still lacking and require further studies. This article gives an overview on critical issues common to all cell culture systems for adherent cells as well as specifics for different types of stem cells in view of small-and large-scale cell expansion and production processes.

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supporting

confidence: 67%

“…120 r.p.m.) [121]. An optimization study using factorial experimental design established an agitation of 100 r.p.m.…”

mentioning

confidence: 99%

Advances in cell culture: anchorage dependence

Merten

2015

Phil. Trans. R. Soc. B

128

103

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“…Spinner flasks or stirred bioreactors have been used for robust expansion of HSCs, because they do not require surface attachment to grow (Zandstra et al 1994;De Léon et al 1998;Ratcliffe et al 2012). Agitation is one of the important parameters in stirred bioreactors or spinner flasks that may cause unexpected cell death (Boehm et al 2010) or differentiation (Hunt et al 2014). There are few reports to evaluate effect of agitation on HSCs differentiation (Jing et al 2013); therefore, here we demonstrate the effect of different agitation speeds of spinner flask on UCB-HSCs expansion and differentiation.…”

Section: Discussionmentioning

confidence: 65%

Agitation increases expansion of cord blood hematopoietic cells and promotes their differentiation into myeloid lineage

2015

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Mechanical stress caused by agitation is one of the factors that can affect hematopoietic stem cell expansion in suspension bioreactors. Therefore, we have investigated the effects of agitation on umbilical cord blood hematopoietic stem cell (UCB-HSC) growth and differentiation. A comparison was made between various agitation rates (20, 40 and 60 rpm) in spinner-flask and cells cultured in glass petri dish as a static culture. Moreover, the fluid dynamic at various agitation rates of spinner-flask was analyzed to determine shear stress. The spinner-flask contained a rotational moving mixer with glass ball and was kept in tissue culture incubator. To reduce consumption of cytokines, UCB-serum was used which widely decreased the costs. Our results determined that, agitation rate at 40 rpm promoted UCBHSCs expansion and their colony forming potential. Myeloid progenitors were the main type of cells at 40 rpm agitation rate. The results of glucose consumption and lactic acid production were in complete agreement with colony assay and expansion data and indicated the superiority of culture in spinner-flask when agitated at 40 rpm over to other agitation speeds and also static culture. Cell viability and colony count was affected by changing the agitation speed. We assume that changes in cell growth resulted from the effect of shear stress directly on cell viability, and indirectly on signaling pathways that influence the cells to differentiate.

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“…Here, we succeeded in overcoming the aggregate size limitation through the break‐up of hiPSC aggregates into small sizes by HA to obtain a high cell density and maintaining pluripotency (Supplementary Figure S3) in suspension culture. Moreover, a higher expansion fold (∼6‐folds higher) was obtained with the break‐up of aggregates by HA, a value higher than those previously reported for hPSC expansion (two‐ to four folds) during long‐term suspension culture (Abbasalizadeh et al, ; Chen et al, ; Hunt et al, ; Olmer et al, ). This successful performance suggests that HA treatment is a promising technique to prevent cell loss during seeding, simplify culture operations without centrifugation or washing, and obtain a high cell density and high growth rate in suspension culture.…”

Section: Discussionmentioning

confidence: 66%

Botulinum hemagglutinin‐mediated in situ break‐up of human induced pluripotent stem cell aggregates for high‐density suspension culture

Nath¹,

Tokura²,

Kim³

et al. 2018

Biotech & Bioengineering

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Large numbers of human induced pluripotent stem cells (hiPSCs) are required for making stable cell bank. Although suspension culture yields high cell numbers, there remain unresolved challenges for obtaining high-density of hiPSCs because large size aggregates exhibit low growth rates. Here, we established a simple method for hiPSC aggregate break-up using botulinum hemagglutinin (HA), which specifically bound with E-cadherin and disrupted cell-cell connections in hiPSC aggregates. HA showed temporary activity for disrupting the E-cadherin-mediated cell-cell connections to facilitate the break-up of aggregates into small sizes only 9 hr after HA addition. The transportation of HA into the aggregates was mediated by transcellular and paracellular way after HA addition to the culture medium. hiPSC aggregates broken up by HA showed a higher number of live cells, higher cell density, and higher expansion fold compared to those of aggregates dissociated with enzymatic digestion. Moreover, a maximum cell density of 4.5 ± 0.2 × 10 cells ml was obtained by aggregate break-up into small ones, which was three times higher than that with the conventional culture without aggregate break-up. Therefore, the temporary activity of HA for disrupting E-cadherin-mediated cell-cell connection was key to establishing a simple in situ method for hiPSC aggregate break-up in bioreactors, leading to high cell density in suspension culture.

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Factorial Experimental Design for the Culture of Human Embryonic Stem Cells as Aggregates in Stirred Suspension Bioreactors Reveals the Potential for Interaction Effects Between Bioprocess Parameters

Cited by 53 publications

References 28 publications

Advances in cell culture: anchorage dependence

Advances in cell culture: anchorage dependence

Agitation increases expansion of cord blood hematopoietic cells and promotes their differentiation into myeloid lineage

Botulinum hemagglutinin‐mediated in situ break‐up of human induced pluripotent stem cell aggregates for high‐density suspension culture

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