January 1, 2015 (Vol. 35, No. 1)

Brian Shapiro, Ph.D. technical writer ATCC
DeZhong Yin, Ph.D. senior scientist ATCC
Anna DiRienzo senior biologist ATCC

Expand Growth by Employing an Optimized Serum- and Feeder Cell-Free Culture System

Recent interest in using induced pluripotent stem cells (iPSCs) for the investigation of disease pathogenesis, as well as drug discovery and development, has resulted in a demand for larger production quantities of these cells.

Early iPSC growth protocols required the presence of mouse-derived fibroblast feeder cells or serum to keep the cells undifferentiated and nourished. However, the use of feeder cells and serum in iPSC culture systems introduces confounding variables into the iPSC production process. These biological products are undefined, raising the possibility of lot-to-lot differences in the product iPSCs. Feeder cells are also difficult to completely remove from these cultures. Therefore, feeder-free and serum-free culture techniques are essential advances for ensuring consistent iPSC production.

An optimized feeder-free and serum-free protocol for iPSC production has been recently created at ATCC. The success of this protocol relies on culturing the iPSCs on a biological matrix, CellMatrix Basement Membrane Gel (CellMatrix; ATCC® ACS-3035), and culturing them in a specialized, defined, serum- and xeno-free stem cell medium, Pluripotent Stem Cell SFM XF/FF (SFMediaXF/FF, ATCC ACS-3002).

In addition to the use of the matrix and medium, the split ratio, culture duration, and handling time of the cells is highly critical during the cell expansion process. Moreover, the expanded cells are well characterized through a variety of methods such as flow cytometrical analysis of pluripotent marker expression, DNA Microarray-based PluriTest analysis of pluripotency, differentiation potential, and karyotyping at early and later passage numbers.

Thus, this iPSC culture system has been highly optimized to ensure that the iPSC cultures retain their undifferentiated state after many passages. In addition, we have validated this culture system for large-scale production of iPSC lines reprogrammed with episomal, retroviral, or Sendai viral methods.


iPSC Expansion and Scale-up

The starting material for this protocol is one cryovial of reprogrammed, undifferentiated iPSCs. Begin by coating two 6 cm culture dishes with CellMatrix at least 1 hour before thawing the iPSCs. When the plates are ready, thaw the cryopreserved iPSCs by resuspending the cells in SFMediaXF/FF containing 10 µM ROCK Inhibitor Y27632 (ATCC ACS-3030).  Pipet the colonies once with a 1,000 µL-tip when resuspending the cell pellet. 24 hours after plating, and every following day, change 100% of the media, removing all cells that do not attach. At this stage, greater than 30 colonies per vial can be expected after 5 days in culture.

To achieve the best results, expand the iPSCs after 5 days in culture, or after they have reached approximately 80% confluence, with more than 90% undifferentiated cells. The split ratio for the cells is typically 1:4. If the iPSCs are not growing sufficiently after 5 days in culture, they may still be used; split the cells at a lower ratio (i.e. 1:2 or 1:3). The iPSC colonies are dissociated from the substrate by incubating them in Stem Cell Dissociation Reagent (ATCC ACS-3020) at 37°C for between 5 and 8 minutes.

At this point, the edges of the colonies should be curling off of the plate. Gently rinse the colonies twice with DMEM: F12 (ATCC 30-2006) to remove all traces of the dissociation reagent. Add SFMediaXF/FF to the plates, scrape the colonies, and then pipet them into a centrifuge tube. After centrifuging the cells, remove all of the supernatant, and resuspend the colonies in SFMediaXF/FF by pipetting colonies three times with a 1,000 µL-tip.

Finally, dilute the colonies to the appropriate ratio and place them on the premade CellMatrix-coated 10 cm dishes (for more detailed information, please refer to the ATCC Stem Cell Culture Guide.

To aid in maintaining the consistency of iPSCs, we recommend that no more than five 10 cm dishes be processed at a time. To increase production, the processing of a second set of five 10 cm dishes may be started halfway through the incubation step of the first set of dishes with the dissociation reagent.

When implementing this larger-scaled production, harvested iPSCs should be placed on ice while processing the other set of dishes. Once production is complete, iPSCs may be plated for use in your experiments, or placed in Serum-free Cell Freezing Medium (ATCC 30-2600) for cryopreservation. We have observed that each 10 cm dish can yield six to eight cryovials of iPSCs containing about 1,000 iPSC colonies per vial. 


Identification and Removal of Differentiated Cells

Undifferentiated iPSCs grow as compact colonies with well-defined borders and, when observed at a high enough magnification, exhibit a high nucleus-to-cytoplasm ratio and prominent nuclei (Figure 1). During the expansion and maintenance of iPSCs, differentiated cells may occur. It is critical to remove these cells early in the workflow to prevent differentiated cells from taking over the cultures and causing a cascade of differentiation. Colonies of differentiated cells exhibit less-defined edges, dark areas, or a non-uniform, loose morphology (Figure 1). Use a fine-tipped aspirating pipette attached to a vacuum source to remove the differentiated cells; ensure that the cultures do not dry out and are not outside the incubator for more than 10 minutes.


Figure 1. Characteristic morphology of iPSCs grown in feeder-free cultures. Phase contrast micrographs of iPSC colonies at A) 4x magnification and B) 10x magnification indicate the compact colony morphology of undifferentiated iPSCs. Arrows indicate well-defined borders and bright areas. Phase contrast micrograph of an iPSC colony undergoing differentiation, C) arrows indicate differentiated cells.

Characterization of iPSCs

Stem cell authentication is often required for journal publication, grant funding, and quality control. ATCC authentication resources and services are available at www.atcc.org/str. We recommend that quality control characterizations such as karyotyping, short tandem repeat analysis, pluripotency, and differentiation potential should be performed after expansion and at least every 15 passages. Immunocytochemistry may be used to assess the pluripotency of iPSC cultures. Undifferentiated iPSCs are characterized by the expression of pluripotent markers such as Tra-1-60, SSEA4, and Tra-1-81 (Figure 2).
Please see Table 1 for QC testing of iPSCs. To quantitate the percentage of undifferentiated iPSCs in culture, flow cytometric analysis of markers of pluripotency and differentiation is performed. An undifferentiated iPSC colony should exhibit high levels (>85%) of Tra-1-60 and SSEA4 and low levels (<15%) of SSEA1 (Table 1).


Figure 2. iPSCs express markers of pluripotency. A) Phase contrast micrograph of iPSCs at 10× magnification. iPSCs stained with antibodies directed against: B) Nanog; C) Tra-1-60; D) overlay of Nanog and Tra-1-60, demonstrating the simultaneous expression of a pluripotency inducer and a marker of pluripotency; E) SSEA4; and F) Tra-1-81. Note: Panels A–D are from the same colony, Panels E and F are from different colonies.

Conclusions

An optimal iPSC culture system has been developed in which iPSC production is increased by replacing the feeder cells and serum with a gel substrate and a complete, serum- and xeno-free medium. In addition, selection of stable iPSC clones and careful maintenance of the cultures, early removal of differentiating cells, and thorough characterization ensures consistent high levels of iPSC production. 



























Brian Shapiro, Ph.D. (bshapiro@atcc.org), is a technical writer, DeZhong Yin, Ph.D., is a senior scientist, and Anna DiRienzo is a senior biologist at ATCC.

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