Cost Effective, Lab-scale iPS Cell Spheroid Culture

[fa icon="calendar"] Feb 9, 2018 10:40:47 AM / by Robert Annand

Many stem cell projects expand to a point where standard culture methods—plates, dishes, flasks, etc.—no longer yield sufficient cells to meet the growing needs for differentiation and regenerative medicine. At this point, larger scale options are required. Traditional bioreactors provide large volume cell culture of liters to hundreds of liters, but they are expensive to buy and expensive to operate. New small scale bioreactors fill the niche between these two extremes perfectly, providing increased cell production over plates or dishes, while being cost-effective to buy and operate.

ABLE 30mL Disposable Bioreactor

ABLE 30mL disposable bioreactor

The ABLE bioreactor system provides standard 30 mL bioreactor (Cat. No. ABBWVS03A-6) and 100 mL bioreactors (Cat. No. ABBWVS10A) containing a unique impeller design to provide low-shear agitation. The vessel itself is made of high-density polycarbonate, and it is surface-treated for biocompatibility to ensure compatibility with stem cell culture. The 30 mL vessel provides a polypropylene cap which is equipped with a filter to allow for passive, sterile gas exchange when the vessels are incubated in a standard cell-culture incubator. The 100 mL flask comes with ports to facilitate exchange of gasses.

 

Magnetic Stir System Configuration

MagStir_Platform_A_background_white

The ABLE Bioreactor Magnetic Stir System (Cat. No. ABBWSS03N0S-6) consists two components. The heat-generating controller module sits outside the incubator and provides for precise, reproducible agitation control. The 6-position stirring module sits inside the incubator, and provides constant, controlled agitation without excessive heat generation, leading to consistent day-to-day and run-to-run performance.

 

Bioreactor Culture of Stem Cells

Shear stress can inhibit the growth of stem cells and, in some cases, promote differentiation3. The unique impellor design of the ABLE Bioreactor is optimized for the culture of stem cells and is designed to minimize the shear stress associated with agitation. The bioreactor allows for the flexibility or using single-cell suspension, passaged colony fragments, or miniopterids for the seed stock, while providing rapid growth to high density. Culture of stem cells in the bioreactor gives uniform spheroid cell clusters with diameters of 200 – 300 μm.

human-ips-spheroids Spheroids of iPSC line 1231A3. Human iPS cells were grown in StemFit medium (Cat No. ASB01) (Ajinomoto) demonstrating the consistency of spheroid sizes after 4 days cultivation in the ABLE 30ml Disposable Bioreactor at 40 rpm.

Stem Cell Growth Curve. Human iPS cells (1231A3) were harvested and dissociated into single cells using TrypLE Select (ThermoFisher), washed and counted.  The single-cells were then seeded at 105 cells/mL in StemFit medium (Cat No. ASB01) supplemented with 10 μM Y27632 (Cat. No. 04-0012) and transferred to the ABLE 30 mL Disposable Bioreactor with constant spinner agitation at 55 rpm.  Cells spheroids were dispersed by TrypLE Select, stained with trypan blue, and counted. 

graph-spheroids-growth

 

Bioreactor-aided differentiation of iPS Cells

In our laboratory, we have adapted our cardiomyocyte differentiation protocol to accommodate the use of the bioreactor. Use of the bioreactor has enabled us to increase the yield of cardiomyocytes four-fold, while reducing medium usage by more than 15%. Adoption of bioreactor culture has also improved the quality of the cardiomyocytes obtained.

Right: Cell yield and medium usage for bioreactor-based differentiation (relative to 2D culture method).

ABLE-cell-yield

Below: Images of cardiomyocyte spheroids differentiated on culture dishes, (left panel), and in the bioreactor (right panel), showing increased cell density in the bioreactor culture.

cardiomyocyte-spheroids-on-culture-dishes1 cardiomyocyte-spheroids-in-ABLE-bioreactor1

 

Summary

The ABLE bioreactor system provides a cost-effective laboratory scale solution for scaling up culture for stem cell projects. The system is engineered to provide consistent results with out worry about shear stress or excess heating. The ABLE bioreactor system also provides an intermediate scale-up option for projects where scale up to industrial culture is desirable, e.g. for emerging regenerative medicine therapies.

 


Note: The ABLE Bioreactor Magnetic Stir System is configured for electrical safety and outlet configurations that are standard in the USA, Canada and Japan.  An accredited European-format equipment configuration is not yet available.

References

  1. Matsuura K. Wada M; Konishi K; Iwamoto U; Sato Y; Tachibana A; Kikuchi T; Iwamiya T; Shimizu T; Yamashita JK; Yamato M; Hagiwara N; Okano T. Fabrication of mouse embryonic stem cell-derived layered cardiac cell sheets using a bioreactor culture system.  PLoS One 2012: 7(12): e52176.
     
  2. Matsuura K. Wada M; Shimizu T; Haraguchi Y; Sato F; Sugiyama K; Konishi K; Shiba Y; Ichikawa H; Tachigbana A; Ikeda U; Yamato M; Hagiwara N; Okano T. Creation of human cardiac cell sheets using pluripotent stem cells. Biochem Biophy Res Com 2012 Aug 24:425(2): 321-327.
     
  3. Toh YC; Voldman J; Fluid shear stress primes mouse embryonic stem cells for differentiation in a self-renewing environment via heparan sulfate proteoglycans transduction. FASEB J 25:1208 (2011).

Topics: Stem Cells, 3D Cell Culture

Robert Annand

Written by Robert Annand

Robert R. Annand, Ph.D.
Senior Technical Product Manager
REPROCELL USA, Inc.