A potentially scalable method for the harvesting of hMSCs from microcarriers

Karen Coopman,Christopher J Hewitt,Qasim A Rafiq,Alvin W Nienow

doi:10.1016/j.bej.2014.02.005

Karen Coopman, Christopher J Hewitt + Show 2 more

Open Access

https://doi.org/10.1016/j.bej.2014.02.005

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Abstract

The use of hMSCs for allogeneic therapies requiring lot sizes of billions of cells will necessitate large-scale culture techniques such as the expansion of cells on microcarriers in bioreactors. Whilst much research investigating hMSC culture on microcarriers has focused on growth, much less involves their harvesting for passaging or as a step towards cryopreservation and storage. A successful new harvesting method has recently been outlined for cells grown on SoloHill microcarriers in a 5L bioreactor [1]. Here, this new method is set out in detail, harvesting being defined as a two-step process involving cell ‘detachment’ from the microcarriers’ surface followed by the ‘separation’ of the two entities. The new detachment method is based on theoretical concepts originally developed for secondary nucleation due to agitation. Based on this theory, it is suggested that a short period (here 7min) of intense agitation in the presence of a suitable enzyme should detach the cells from the relatively large microcarriers. In addition, once detached, the cells should not be damaged because they are smaller than the Kolmogorov microscale. Detachment was then successfully achieved for hMSCs from two different donors using microcarrier/cell suspensions up to 100mL in a spinner flask. In both cases, harvesting was completed by separating cells from microcarriers using a Steriflip® vacuum filter. The overall harvesting efficiency was >95% and after harvesting, the cells maintained all the attributes expected of hMSC cells. The underlying theoretical concepts suggest that the method is scalable and this aspect is discussed too.

Highlights

There is significant emphasis on improving human mesenchymal stem cell expansion techniques due to the interest in these cells for treating a number of diseases/disorders [2]
In a recently-published paper [1], we have reported the successful cultivation of human mesenchymal stem cell (hMSC) on Plastic 102-L microcarriers (SoloHill, USA) in a 5 L bioreactor (2.5 L working volume) agitated at an impeller speed, NJS, just sufficient to keep all the microcarriers in suspension
There is little focus on harvesting in those studies reported in the literature investigating the expansion of hMSCs on microcarriers

Summary

Introduction

There is significant emphasis on improving human mesenchymal stem cell (hMSC) expansion techniques due to the interest in these cells for treating a number of diseases/disorders [2]. It is important to recognise that, independent of whether the cells are to be reused for further expansion following a passage or stored prior to use for therapeutic or other purposes, harvesting involves two steps. The term ‘detachment’ or ‘dissociation’ is used just for the first step of removing the cells from the microcarrier surface; and the second step is designated ‘separation’ and involves the use of techniques such as filtration or centrifugation. These issues will only be exacerbated as expansion scale increases and it is crucial to consider cell harvesting strategies from the outset so as to ensure a viable, complete bioprocess

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