Pall Xpansion® bioreactor supports progenitor cell growth to >1 million cells/cm² and proper cell differentiation



Andrew Laskowski and Siddharth Gupta




Problem Statement


Several cell therapies approved by the FDA or currently in clinical development rely on the expansion of adherent primary cells. The current platforms used to expand these cells are labor-intensive, consist of many open handling steps, have a large manufacturing footprint, and do not provide adequate process control.


Gap Addressed by Pall


The need for a GMP-compliant manufacturing platform for adherent human cells that can be implemented in research labs, allowing for control of the cellular environment, and can be easily scaled up for Phase II, Phase III, and commercial manufacturing


Proposed Solution


Pall’s Xpansion single-use bioreactor offers a scalable manufacturing platform with online monitoring and control of critical quality attributes (CQAs) for cell therapy applications


Experimental Goals


  • Successfully demonstrate Xpansion bioreactor plate coating with a proprietary protein solution that enhances cell attachment
  • Expand and differentiate a previously untested proprietary progenitor cell in the 10-plate Xpansion bioreactor (Xpansion 10) using a 16-day and 22-day differentiation protocol. Compare results to T-150 controls
  • Optimize the procedure to harvest and recover cells from the Xpansion bioreactor
  • Develop a post-harvest cell concentration and washing process to handle the larger cell suspension volumes from the Xpansion 10 bioreactor


Success Criteria


  • 2-4x amplification of cells and equivalent harvest cell densities to the control
  • >95% viability post cell washing – >90% differentiation to a more mature cell fate
  • Microscopic evidence of cellular self-assembly into characteristic monolayer with cell colony formation




Schematic Diagram of Xpansion Bioreactor Components




  • Single-use multi-plate bioreactor available in 4 sizes (10, 50, 100, and 200 plates; 612 cm2 per plate)
  • Cell attachment surface is a circular, cell culture-treated polystyrene plate (A)
  • Stacking of pates creates a center column containing a coil of gas tubing for CO2 and O2 exchange (B)
  • Plates have radial slits and are stacked such that these slits are staggered, forcing media flow uniformly over the plate surface (C)
  • Mixing is achieved via an impeller located under the bottom plate (D)
  • Constant, low shear stress maintained by keeping plate distance, geometry, and media mixing velocity constant
  • Head plate contains single-use pH/DO PreSens♦ sensors and sampling port
  • Two large liquid addition and removal lines at bottom of reactor (E)








Cell Concentration, Wash, and Fill 







1. Harvest Optimization Studies








  • A TrypLE-based harvest procedure was optimized in T-150 flasks by testing varying dilutions, incubation temperatures, and incubation times to maximize cell recovery and minimize cell aggregation
  • 100% TrypLE and incubation temperatures of 37 °C (test case A2, A6, and A8) generally resulted in high cell recoveries and less cell clumping
  • Incubation time did not have an impact: 55 minutes preferred to reduce processing time


2. Cell Recovery, Viability, and Morphology





Run 1


  • Cell density post-wash is less than estimated cell density pre-harvest and provides opportunities for improvement in the downstream operations:
    • ~7% cell loss in the spent media due to premature cell detachment
    •  ~7% of cells did not completely detach from the Xpansion 10 bioreactor and were instead collected in a subsequent harvest step
    • Cell loss in Xpansion 10 bioreactor compared to control due to longer centrifugation process and cell aggregation before straining
    • Cells progressively aggregated during counting, leading to a lower cell count average
    • With an optimized process, 4.6 million cells/cm2 or a 6x amplification (close to ‘gold standard’), would have been recovered from Xpansion 10 bioreactor
  • Cell density and viability in the Xpansion 10 bioreactor otherwise equivalent to the flatware control
  • Cells self-assembled into a characteristic monolayer with cell colony formation in the Xpansion 10 bioreactor, similar to the flatware controls


Run 2


  • Cell density and viability in Xpansion 10 bioreactor equivalent to controls
  • Cells expanded less than Run 1, likely due to the differences between the two differentiation protocols
  • Less cell aggregation and yield loss occurred post-bioreactor harvest. The automated cell washing process was more effective than centrifugation at concentrating cells and maintaining a single-cell suspension





3. Cell Differentiation





  • Population level cell surface marker expression was equivalent between Xpansion 10 bioreactor and controls for both Run 1 and Run 2
  • SM1 was expressed in >90% of the cell population, as indicated by ICC, for both Run 1 and Run 2
  • The improvement from Run 1 to Run 2 was due to changes between the 16-day vs. 22-day differentiation protocols




  • The Xpansion bioreactor plates were successfully coated with a proprietary protein critical to cell attachment and proliferation
  • The Xpansion bioreactor can support adherent human cell culture at densities of >1 million cells/cm2 for extended culture durations
  • T-flasks can be used to optimize several harvest parameters and the optimized parameters translate successfully when transferred to the Xpansion 10 bioreactor
  • Using two different protocols, the Xpansion bioreactor facilitated 3-4x amplification of cells and >95% cell viability. This was equivalent to flatware controls
  • Cells self-assembled into a characteristic monolayer with cell colony formation in the Xpansion 10 bioreactor, similar to the flatware controls
  • Cells differentiated to the more mature cell fate equivalently in both the Xpansion 10 bioreactor, and the controls, achieving >90% differentiation using both differentiation protocols
  • The automated washing process was more effective at concentrating and washing larger volumes of cell suspension compared to centrifugation, reducing both cell aggregation and yield loss


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