Stem cells 2013 July-19

 

Cryopreservation of pluripotent stem cell aggregates in defined protein-free formulation.

Biotechnol Prog. 2013 Jan-Feb;29(1):143-53.

Sart S, Ma T, Li Y.

Abstract

Background:

Cultivation of undifferentiated pluripotent stem cells (PSCs) as aggregates has emerged as an efficient culture configuration, enabling rapid and controlled large scale expansion.  Aggregate-based PSC cryopreservation facilitates the integrated process of cell expansion and cryopreservation, but its feasibility has not been demonstrated.

Objective:

The goals of current study are to assess the suitability of cryopreserving intact embryonic stem cell (ESC) aggregates and investigate the effects of aggregate size and the formulation of cryopreservation solution on ESC survival and recovery.

Results:

The results demonstrated the size-dependent cell survival and recovery of intact aggregates.  In particular, the generation of reactive oxygen species (ROS) and caspase activation were reduced for small aggregates (109 ± 55 µm) compared to medium (245 ± 77 µm) and large (365 ± 141 µm) ones, leading to the improved cell recovery.  In addition, a defined protein-free formulation was tested and found to promote the aggregate survival, eliminating the cell exposure to animal serum.  The cryopreserved aggregates also maintained the pluripotent markers and the differentiation capacity into three-germ layers after thawing.

Conclusion:

In summary, the cryopreservation of small PSC aggregates in a defined protein-free formulation was shown to be a suitable approach towards a fully integrated expansion and cryopreservation process at large scale.

 

Supplementary:

Production of therapeutic cells from pluripotent stem cells (PSCs) starts from thawing cells from qualified cell banks such as Master Cell Bank (MCB) or Working Cell Bank (WCB) under current good manufacturing practices.  The quality of cell bank plays an important role in PSC bioprocessing because it impacts post-thaw expansion, the efficiency of subsequent specific lineage differentiation, and process reproducibility.  A robust cryopreservation and thaw process in generating MCB and WCB thus become the crucial first step that ensures the quality of PSC-based cell products.

Recently, undifferentiated PSC aggregates have been shown as efficient cell organization for large scale expansion in suspension culture, but the feasibility of the aggregate-based cryopreservation has not been investigated.  The results of the present study revealed the role of cellular microenvironment in the aggregate-based cryopreservation and a size-dependent PSC survival and recovery due to the expression of reactive oxygen species (ROS) and caspase (Figure 1), demonstrating the feasibility of aggregate-based cryopreservation for an integrated expansion and cryopreservation process in a defined protein-free formulation.

yan li

Figure 1. Generation of reactive oxygen species (ROS) and caspase activation in the aggregates immediately post thaw.  (A) Fluorescent images of ROS expression under light.  Scale bar: 100 μm.  (B) The percentage of ROS positive aggregates.  (C) Fluorescent images of caspase expression.  Scale bar: 200 μm.  Long arrow: aggregates partially stained with caspase; short arrow: aggregates totally stained with caspase.  (D) The percentage of caspase positive aggregates.  N.D.: not detectable.  * p-value <0.05.  IA: intact aggregate. S: small; M: medium; L: large. TBHP: tert-butyl hydroperoxide.

This figure is adapted from the referred paper:

Sebastien Sart, Teng Ma, Yan Li.  Cryopreservation of pluripotent stem cell aggregates in defined protein-free formulation. Biotechnology Progress. 2013; 29: 143-153.

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