One of the main problems in clinical application of PSCs is the high cost associated with their production, with recombinant growth factors representing the most expensive aspect of medium formulation for PSC culture. To save growth-factor usage, strategies have been employed to retain the accumulated growth factors by using dialysis-based culture systems. However, most of these large culture systems are not well optimised to push their potential to enable low-cost expansion by increasing cell density (i.e., ≥ 1 × 107 cells/mL)25. High-density culture might also increase additional risks, such as high mechanical stress due to aggregate collision18 and excess agglomeration17. To address this problem, we designed a miniaturised dialysis-culture system for first-line evaluation of high-density culture. Our findings showed that this system was able to support the production of hiPSCs in high-density volumetric yield up to ~ 32 × 106 cells/mL and with decent pluripotency preservation and differentiation capability. Additionally, adequate hiPSCs were acquired by reductions in mechanical stress and excess agglomeration via the addition of FP003 biopolymer solution. The increase in final cell density might allow a reduced requirement for expensive growth factors8,9. The dialysis culture mimicked in vivo conditions, where the production of autocrine factors is utilised in compact, three-dimensional tissue receiving a continuous supply of nutrition from the blood. Mimicking this phenomenon in an in vitro system allows acquisition of healthy and high-quality PSCs in a cost-effective manner through maintenance of their autonomous homoeostasis in high-density cell culture. This culture system offers new insight into utilisation of exogenous and endogenous growth-factor accumulation in a cost effective dialysis-culture system (Supplementary Fig. 2).
We observed successful accumulation of several exogenous growth factors in the dialysis-culture medium to support further proliferation. We used the simple medium formulation developed by Chen et al.26 and based on the minimum components required for PSC culture, including Dulbecco’s modified Eagle medium (DMEM)/F12 basal medium and supplementation with insulin, transferrin, TGF-β1, and FGF2 (Table 1)27. Similar to that used for other PSC cultures, this medium includes a large amount of FGF228 and insulin29, whereas TGF-β1 is often supplemented in small amounts to support the pluripotency30. A previous study reported that some autocrine factors, such as FGF231–33, TGF-β134–36, and insulin-like growth factors37–39, are secreted by stem cells to support their homoeostasis, which is reinforced by the addition of exogenous FGF2 and insulin to the culture medium. However, the remaining excess growth factors, such as insulin and FGF2, are usually removed when the medium is replaced. Therefore, utilisation of accumulated insulin and FGF2 by dialysis culture might further reduce costs. Additionally, TGF-β1 is significantly depleted over time in dialysis culture, making it insufficient to support high-density hiPSCs culture. Although recent studies show that PSCs can still proliferate and maintain their pluripotency in the absence of TGF-β1 and FGF240,41, PSCs cultured in their absence are less dependent on glycolytic pathways, which reduces their proliferation40. By contrast, in the present study, we showed that gene expression associated with pluripotency remained enhanced, suggesting that support might come from other autonomous regulatory pathways.
The endogenous growth factors secreted by hiPSCs affect pluripotency maintenance and proliferation during high-density expansion. Previous reports show that better cellular metabolism and yield can be achieved by increasing inoculation density42. Increases in cell density might also play an important role in maintaining pluripotency through improved accumulation of endogenous growth factors and their autocrine derivatives. To confirm their advantageous accumulation, we chose NODAL as a selected candidate factor to represent the accumulation of important hiPSC autocrine factors. NODAL and TGF-β1 levels are mediated by a similar mechanism via SMAD2/3 signalling to maintain PSC pluripotency by enhancing NANOG upregulation to maintain balanced differentiation into an ectoderm or endoderm lineage (Fig. 7)22,43−47. In response to these mechanisms, we found that NODAL levels increased in a density dependent manner and consistently correlated with NANOG gene-expression patterns. This indicated that NODAL might reconstitute the role of TGF-β1 in maintaining pluripotency and retaining differentiation potential into three human embryonic germ layers cell type.
As expected, medium refinement by the dialysis-culture system successfully supported further hiPSC proliferation. To compensate for the accumulation of toxic metabolic products resulting from cellular metabolism, small molecules, such as lactate, need to be frequently removed from the culture medium when performing high-density culture. Continuous lactate removal using dialysis fed-batch support successfully maintained lactate concentrations in the upper culture compartment below the critical concentration, thereby eliminating the growth-limiting conditions caused by lactate accumulation while supplying glucose. Moreover, we observed an exponential growth curve associated with the maximum cell density and its relationship with lactate concentration, suggesting that there remains potential for further increases in cell density for this system. Interestingly, a significant amount of glucose remained, even without use of the dialysis culture and in the absence of medium replacement, from the first day of culture, although the cell number was extremely depleted relative to the early days of the culture. This suggested that lactate accumulation rather than glucose starvation was the primary limiting factor for cell proliferation in suspension culture. This phenomenon occurs, because hiPSCs exhibit higher anaerobic respiration and metabolism relative to their differentiated cell types. Consequently, lactate secretion was much higher than glucose consumption due to the dependency of PSCs on glycolysis for their energy demands48.
Addition of the FP003 biopolymer successfully created a low-shear-stress culture environment and prevented hiPSC agglomeration. PSCs exhibit a high tendency to aggregate when cultured in suspension49; therefore, we utilized dynamic conditions to control aggregate size and prevent excess agglomeration2,49−52. The lack of agitation can result in large aggregates with necrotic areas often caused by unequal exposure to nutrition and secreted toxic metabolites due to mass-transfer limitations and failure to reach some aggregated cells53. However, the excessive mechanical stress resulting from this condition can also potentially induce unwanted spontaneous differentiation and affect cellular viability51,54. To prevent the negative effect of this agitation, we added FP003 to the culture medium. Otsuji et al.17 revealed the potential of FP003 for preventing agglomeration in static hiPSC suspension culture. Interestingly, addition of FP003 did not significantly affect the transfer of micromolecules, such as glucose and lactic acid, in our dialysis-culture system. Additionally, comparison with rotary suspension culture indicated that FP003 significantly decreased cellular injury, possibly due to increased culture-medium viscoelasticity via modification of rheological properties. In suspension culture, low-acyl gellan gum in FP003 forms a microfiber-network structure that keeps cells floating in a well-dispersed manner and blocks agglomeration between aggregates (Fig. 1B). As a result, the high-density expansion resulted in a uniform aggregate population with decent growth in size. Moreover, the 4-day culture grew to a tolerable size that allowed the transfer of oxygen, nutrition, and waste products throughout the aggregates. This condition was also confirmed by the absence of necrotic areas inside the aggregates.
This smaller-scale dialysis-culture platform would be useful for evaluating the feasibility of dialysis operations in PSC culture using minimal resources. The complexity of currently available large-scale culture systems makes technical operations during PSC culture challenging to optimise. Additionally, medium refinement could overcome the requirement for a high metabolic rate demanded by PSCs in the forms of nutrition transfer, waste-product removal, and growth-factor supplementation. This culture system allows various culture conditions simultaneously, enabling optimization of parameters, such as cell metabolism, growth-factor use, device permeation associated with cell growth, pluripotency, and differentiation capacity. Moreover, mathematical simulations describing the metabolic kinetics associated with both exogenous and endogenous growth factors represent useful references when designing larger-scale dialysis-culture systems. However, larger-scale systems, such as slow-turning lateral vessels8 or stirred-suspension bioreactors with dialysis support9, might still have several problems related to oxygenation and mechanical stress that are broadly design dependent. Although this study was limited to the proliferation phase, the potential reductions in production costs associated with utilisation of both endogenous and exogenous growth factors might contribute to future optimisation of larger-scale and cost-effective hiPSC-production methods. Furthermore, such methods could increase PSC proliferation and differentiation, which require optimal growth-factor and autocrine utilisation.
In summary, we described a dialysis-culture system that promotes efficient hiPSC expansion at a high density while maintaining their pluripotency and differentiation capacities by facilitating growth-factor accumulation together with important autocrine factors under a low-hydrodynamic-stress culture environment. This study provides novel insight into the feasibility of minimum growth-factor usage, which might significantly promote cost reductions in dialysis-based PSC production.