A miniature dialysis-culture device allows high-density human-induced pluripotent stem cells expansion from growth factor accumulation

Abstract

Three-dimensional aggregate-suspension culture is a potential biomanufacturing method to produce a large number of human induced pluripotent stem cells (hiPSCs); however, the use of expensive growth factors and method-induced mechanical stress potentially result in inefficient production costs and difficulties in preserving pluripotency, respectively. Here, we developed a simple, miniaturized, dual-compartment dialysis-culture device based on a conventional membrane-culture insert with deep well plates. The device improved cell expansion up to approximately ~3.2 to 4×107 cells/mL. The high-density expansion was supported by reduction of excessive shear stress and agglomeration mediated by the addition of the functional polymer FP003. The results revealed accumulation of several growth factors, including fibroblast growth factor 2 and insulin, along with endogenous Nodal, which acts as a substitute for depleted transforming growth factor-β1 in maintaining pluripotency. Because we used the same growth-factor formulation per volume in the upper culture compartment, the cost reduced in inverse proportional manner with the cell density. We showed that growth-factor-accumulation dynamics in a low-shear-stress environment successfully improved hiPSC proliferation, pluripotency, and differentiation potential. This miniaturised dialysis-culture system demonstrated the feasibility of cost-effective mass production of hiPSCs in high-density culture.

Generation of high-density human induced pluripotent stem cell derived-liver organoid by enabling growth factors accumulation in a simple dialysis medium refinement culture platform

“…To address this problem, we include the gellan gum-based-biopolymer FP001 to reduce the mechanical stress and excess agglomeration while enabling e cient high-density culture at the same time 17,18 . Based on our previous study 7 , the application of this biopolymer has shown its potential in protecting the high-density hiPSCs aggregates from mechanical stress induced-apoptosis and necrosis, which may negatively affect cell proliferation and cell quality. In this study, we con rmed that the inclusion of this biopolymer has shown its potential to improved the HLCs production (Supplementary Fig.…”

“…A physical condition, such as excessive mechanical stress may signi cantly affect the differentiation e ciency into the hepatic lineage. Although a gentle dynamic culture system has been known to improve the hepatic differentiation 12,13 , the mechanical stress coming from hydrodynamic mixing and aggregates collision in rotational culture may negatively interfere with the differentiation process 7 . The previous study by Vosough et al was shown that excessive shear stress can reduce the hepatic differentiation e ciency during culture in stirred tank bioreactor 14 .…”

“…This culture system was rstly utilized to produce the monoclonal antibody from hybridoma cell lines in a high cell density 9,10 . Our previous study has shown their potential in the highly e cient production of undifferentiated hiPSCs by the accumulation of important growth factors required for maintaining pluripotency and proliferation; while simultaneously maintaining the medium re nement in a very high cell density 7,11 . Since the differentiation process required a larger amount of these expensive factors, the cost reduction may be signi cantly enhanced by using this dialysis device.…”

“…Nutrition supply, waste metabolic byproduct removal, and mechanical-related stress becoming the main challenges that must be overcome in the current hiPSCs culture. Additionally, the use of growth factors caused by daily medium removal can lead to high production costs and ine cient differentiation processes [6][7][8] . The dialysis culture system is one of the potential methods that can be utilized for improving differentiation e ciency.…”

Strategies for Generating Human Pluripotent Stem Cell- Derived-Organoid Culture for Disease Modelling, Drug Screening, and Regenerative Therapy

“…Since the usage of cytokine or growth factors still becoming a necessity, an alternative solution is developed to optimize the usage of growth factors efficiency during differentiation. For example, using a specific molecular weight cutoff dialysis membrane barrier to recycle and accumulate the high molecular weight endogenous and exogenous growth factors while continuously maintaining the exchange between the small molecular weight nutrition and toxic metabolic byproducts [83]- [86]. This approach may significantly improve the production efficiency per unit of hPSCs-derived organoids.…”