Culture medium refinement by dialysis for the expansion of human induced pluripotent stem cells in suspension culture

Abstract

Human induced pluripotent stem cells (hiPSCs) secrete essential autocrine factors that are removed along with toxic metabolites when the growth medium is exchanged daily. In this study, after determining the minimum inhibitory level of lactic acid for hiPSCs, a medium refining system was constructed by which toxic metabolites were removed from used culture medium and autocrine factors as well as other growth factors were recycled. Specifically, about 87 % of the basic fibroblast growth factor and 80 % of transforming growth factor beta 1 were retained in the refined medium after dialysis. The refined medium efficiently potentiated the proliferation of hiPS cells in adherent culture. When the refining system was used to refresh medium in suspension culture, a final cell density of (1.1 ± 0.1) × 10 cells mL was obtained, with 99.5 ± 0.2 % OCT 3/4 and 78.3 ± 1.1 % TRA-1-60 expression, on day 4 of culture. These levels of expression were similar to those observed in the conventional suspension culture. With this method, culture medium refinement by dialysis was established to remove toxic metabolites, recycle autocrine factors as well as other growth factors, and reduce the use of macromolecules for the expansion of hiPSCs in suspension culture.

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

“…Cells were incubated at 37 °C in a humidified atmosphere with 5% CO 2 , and the medium was changed daily with fresh one. On day 4, after reaching confluence, cells were subcultured as described elsewhere (Nath, Nagamori, Horie, & Kino‐oka, ). Briefly, hiPSCs were treated with 5 mM ethylenediaminetetraacetic acid/phosphate‐buffered–saline (PBS) with 10 µM ROCK inhibitor for 7 min incubation at room temperature.…”

“…To investigate the effect of aggregate break‐up by HA, hiPSCs were detached from the culture substrate as described elsewhere (Nath, Nagamori, et al, ). After dissociating the hiPSC colony into single cells, 0.1 × 10 6 cells ml −1 were seeded with 10 µM ROCK inhibitor in a 30 ml bioreactor.…”

“…In the control culture, aggregates were not treated with HA or not broken into small sizes, and cultured for an additional 96 hr. The culture medium was changed with fresh medium at frequent intervals to reduce the toxic effect of lactic acid as high concentrations of lactic acid reduce the growth and pluripotency of hiPSCs in suspension culture (Nath, Nagamori, et al, ).…”

“…Cells were collected at the final day of culture (at t = 96 or 192 hr) to assess the expression of the pluripotency markers OCT 3/4 and SSEA 4 by flow cytometry. The flow cytometry procedure was similar to that described previously (Nath, Nagamori, et al, ). Briefly, single cells were prepared, treated with a Cytofix/Cytoperm™ permeabilization kit (BD Biosciences, San Jose, CA), and incubated with primary antibody (mouse monoclonal OCT 3/4, Santa Cruz Biotechnology, Dallas, TX), and secondary antibody (APC anti‐mouse IgG, BD Biosciences).…”

Bioprocess Design Considerations for Cultured Meat Production With a Focus on the Expansion Bioreactor

“…Potential downstream units may include one or more of membrane filtration, (electro)dialysis, precipitation, solvent extraction, and adsorption. Relevant industrial examples include fermentation (Li et al, 2008;Lee et al, 2017;Seankham et al, 2017); water treatment (Bódalo et al, 2005); and iPSC culture (Nath et al, 2017). Further product formulation units capable of chopping, drying, flavoring, texturizing, packaging and labeling may be required (Park et al, 2014) depending on the product of interest.…”