Induced Pluripotent Stem Cells on a Chip: A Self-Contained, Accessible, Pipette-less iPSC Culturing and Differentiation Kit

Abstract

Over the past decade, induced pluripotent stem cells (iPSCs) have become a major focus of stem cell and developmental biology research, offering researchers a clinically relevant source of cells that are amenable to genetic engineering approaches. Though stem cells are promising for both research and commercial endeavors, iPSC-based assays require tedious protocols that include complex treatments, expensive reagents, and specialized equipment that limit their integration into academic curricula and cell biology research groups. Expanding on existing Kit-On-A-Lid-Assay (KOALA) technologies, we have developed a self-contained, injection molded, pipette-less iPSC culture and differentiation platform that significantly reduces associated costs and labor of stem cell maintenance and differentiation. The KOALA kit offers users the full range of iPSC culture necessities, including cell cryopreservation, media exchanges, differentiation, endpoint analysis, and a new capability, cell passaging. Using the KOALA kit, we were able to culture ~20,000 iPSCs per microchannel for at least 7 days, while maintaining stable expression of stemness markers (SSEA4 and Oct4) and normal iPSC phenotype. We also adapted protocols for differentiating iPSCs into neuroepithelial cells, cardiomyocytes, and definitive endodermal cells, a cell type from each germ layer of human development.

Keywords: cell culture incubators; lab-on-a-chip; microfluidics; microtechnology.

Figure 1 -

KOALA components and diagram. (A) Koala base. (B) KOALA lid. (C) KOALA cryopreservation lid. (D) Diagram of how KOALA operates: (1) KOALA lid as placed on top of the KOALA base so the media well and absorbent pad make contact with the inlet and outlet posts. (2) Media flows into the microchannel from the media well due to capillary action. (3) Media continues to flow due to capillary action from the absorbent pad. (4) Once all of the media flows out of the lid, it will “pin” inside the microchannel, which causes a “break” in contact between the fluid in the microchannel and the absorbent pad. (5) The lid is removed and the new media/reagent remains in the KOALA base.

Figure 2 -

iPSC culture in KOALA versus well plate. (A) Seeding densities versus viability rate in KOALA microchannels. (B) Total number of adhered iPSCs over time in Matrigel or vitronectin-coated KOALA microchannels. (C) Total number of adhered iPSCs over time in KOALA microchannels versus well plates. (D) Total number of adhered iPSCs per culture area over time in KOALA microchannels versus well plates. (E) Total number of adhered iPSCs per culture volume over time in KOALA microchannels versus well plates. (F) Percent of Oct4⁺/SSEA4⁺ cells over time in KOALA microchannels versus well plates. (G) Fluorescent imaging of IMR90–4 iPSCs after 2 days in a well plate (top) or KOALA microchannel (bottom) immunostained for SSEA4 (green), OCT4 (red), and a nuclear stain (blue). Graphs depict average ± standard deviation across 12 technical and 3 biological replicates. *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 3 -

Cryopreservation lid characterization. (A) Total adhered cells in KOALA microchannels on days 2 and 5 after seeding using the KOALA cryopreservation lid or by manual seeding with pipetman. No statistical significance was determined between the KOALA cryopreservation lid and manual seeding with pipetman. (B) RT-qPCR of iPSCs after 5 days of cell culture in a KOALA microchannel after seeding using the KOALA cryopreservation lid or by manual seeding with pipetman. No statistical significance was determined between the KOALA cryopreservation lid and manual seeding with pipetman. Graphs depict average ± standard deviation.

Figure 4 -

KOALA passaging device characterization. (A) Diagram of KOALA passaging device operation. (B) Image of passaging device filled with media. (C) Graph of percentage of media transferred to new KOALA microchannel as a function of media reservoir volume. Includes theoretical maximum (gray line). (D) Percentage of cells transferred to new KOALA microchannel as a function of media reservoir volume. Graphs depict average ± standard deviation.

Figure 5 -

Directed differentiation in KOALA. (A) Neuroepithelial differentiation. (i) RT-qPCR of 2-, 5-, and 7-day differentiated IMR90–4 iPSCs, assaying for PAX6, SOX2, and POU5F1. (ii) Fluorescent image of differentiated IMR90–4 iPSCs, staining for Pax-6 (red) and N-cadherin (green). (B) Cardiomyocyte differentiation. (i) RT-qPCR of 2-, 5-, and 7-day differentiated IMR90–4 iPSCs, assaying for T, ISL1, GATA4, and POU5F1. (ii) Fluorescent image of differentiated IMR90–4 iPSCs, staining for Isl-1 (red) and cTnT (green). (C) Definitive endoderm differentiation. (i) RT-qPCR of 2-, 5-, and 7-day differentiated IMR90–4 iPSCs, assaying for FOXA2, SOX17, and POU5F1. (ii) Fluorescent image of differentiated IMR90–4 iPSCs, staining for SOX17 (red) and FoxA2 (green). Graphs depict average ±standard deviation.