Conclusions
- Addition of Recombinant Insulin significantly improves iPSC morphology and proliferation.
- The presence of Recombinant Insulin substantially enhances cell attachment and maintains unaltered pluripotency potential.
- Optimizing insulin concentration for iPSC cultures is crucial for enhancing iPSC cultures with tailored conditions, a prerequisite for successful scaling-up..
Background
The development of induced pluripotent stem cell (iPSC) technology has revolutionized cell therapy and regenerative medicine applications. iPSCs possess the potential for virtually unlimited expansion and are capable of differentiating into a wide range of somatic cell types.
Consistent production of high-quality iPSCs is dependent on intricate optimizations of cell culture conditions, which have been thoroughly investigated to identify the optimal formulation that can support proliferation while preserving differentiation potential. Insulin is one of the eight components identified to be essential for the quality of iPSC cell cultures.
In this study, we show the benefits of adding optimized concentrations of Recombinant Insulin to chemically-defined cell culture media for iPSC cell cultures.
Study description
In the study, iPSCs were cultured in a commercially available chemically-defined (CD) media, which contained 7 out of 8 essential cell culture supplements required for efficient iPSC growth, with the missing supplement being insulin. Various concentrations of Recombinant Insulin (0, 5, 10 and 20 mg/L) were then added to the cell culture media to assess its impact on iPSC cell morphology, proliferation, cell attachment, and expression of pluripotency markers.
Daily cell counts and morphology observations were conducted until Day 5. To evaluate the attachment of cells seeded as single cells or aggregates, the cells were detached using either Accutase or ReLeSR, respectively. Subsequently, the cells were cultured in the CD media, supplemented with 5 μM of ROCK inhibitor (only for single-cell passaging). The culture media was then supplemented with either 10 mg/L Recombinant Insulin or without it, and cell attachment was assessed by staining the cells with crystal violet.
To evaluate the stability of iPSCs cultured in the presence of Recombinant Insulin, the expression of pluripotency markers (Oct3/4 and Tra-1-81) was measured by flow cytometry after one passage or after five passages (25 days in total).
Results
iPSCs cultured in CD media without insulin exhibited slow growth and an irregular, spiky morphology, suggesting cellular stress. In contrast, iPSCs cultured in the presence of Recombinant Insulin displayed colonies with dense cellular packing and well-defined borders, even at a concentration as low as 5 mg/L (Fig. 1A). Furthermore, iPSC proliferation in CD media supplemented with Recombinant Insulin was notably higher compared to proliferation without insulin (Fig. 1B), indicating the crucial role of insulin in maintaining healthy and proliferating iPSC cultures.
Additionally, the presence of Recombinant Insulin significantly enhanced the attachment of iPSCs to the culture well, irrespective of whether the cells were seeded as single cells or aggregates (Fig. 2). Lastly, the expression of pluripotency markers remained unchanged (>95%) after the addition of Recombinant Insulin to the culture media, following both a single passage and five passages. This suggests that Recombinant Insulin does not impact the stability of iPSCs in culture, preserving the cells in an undifferentiated state (Fig. 3).
Figure 1A:
Figure 1B:
Figure 1: Recombinant Insulin improves iPSC cell morphology and proliferation. (A) Representative images of iPSC morphology in different insulin conditions analyzed through brightfield microscopy on days 3-4 from one out of three biological runs. Scale bar = 500 μm. (B) Total cell count at various concentrations of insulin, measured over 5 days. Data represents mean cell count across three biological runs ± SEM.
Figure 2:
Figure 2: Recombinant Insulin improves iPSC attachment to the cell culture well. Representative images of iPSCs attachment in different seeding conditions, aggregate (top panel) and single cell (bottom panel), with or without 10 mg/L insulin. Cell attachment analyzed through crystal violet dye staining analyzed 24 hours after seeding from one out of three biological runs. Scale bar = 500 μm.
Figure 3:
Figure 3: Recombinant Insulin does not affect stability of iPSC culture. Average pluripotency markers (Tra-1-81 and Oct 4) expression after one passage and five passages (25 days) in iPSCs cultured without insulin or with 10 mg/L of insulin, in 3 biological runs ± SEM. Pluripotency is represented as % of cells expressing the Tra-1-81 and Oct 4 pluripotency markers.
