Agitation conditions for the culture and detachment of hMSCs from microcarriers in multiple bioreactor platforms

Alvin W Nienow,Christopher J Hewitt,Thomas R.J Heathman,Veronica A.M Glyn,Gonҫalo N Fonte,Mariana P Hanga,Karen Coopman,Qasim A Rafiq

doi:10.1016/j.bej.2015.08.003

Abstract

In our recent work in different bioreactors up to 2.5L in scale, we have successfully cultured hMSCs using the minimum agitator speed required for complete microcarrier suspension, NJS. In addition, we also reported a scaleable protocol for the detachment from microcarriers in spinner flasks of hMSCs from two donors. The essence of the protocol is the use of a short period of intense agitation in the presence of enzymes such that the cells are detached; but once detachment is achieved, the cells are smaller than the Kolmogorov scale of turbulence and hence not damaged. Here, the same approach has been effective for culture at NJS and detachment in-situ in 15mL ambr™ bioreactors, 100mL spinner flasks and 250mL Dasgip bioreactors. In these experiments, cells from four different donors were used along with two types of microcarrier with and without surface coatings (two types), four different enzymes and three different growth media (with and without serum), a total of 22 different combinations. In all cases after detachment, the cells were shown to retain their desired quality attributes and were able to proliferate. This agitation strategy with respect to culture and harvest therefore offers a sound basis for a wide range of scales of operation.

Highlights

Unlike cell culture for traditional biopharmaceutical production, where the product of interest is usually a recombinant protein, cells expanded on microcarriers for cell therapies form the basis of the therapeutic
Spinner flasks were used for culture followed by detachment in the same vessel whilst in Case 17, it was established that the DASGIP bioreactor could be used for both steps
The assessment of harvest efficiency was based firstly on a count of the cells on the microcarriers after culture followed by visual observation of the suspension after detachment by intense agitation, which indicated that the microcarriers no longer had cells attached to them and that they were present in suspension as single cells

Summary

Introduction

Unlike cell culture for traditional biopharmaceutical production, where the product of interest is usually a recombinant protein, cells expanded on microcarriers for cell therapies form the basis of the therapeutic. Successful cell recovery is determined by quantities of cells recovered and by the quality of recovered cells. Effective cell recovery will reduce overall cost of goods by increasing process efficiency and enabling process intensification. Few studies have harvested greater than millilitre samples of the microcarrier culture. This lack of publications on harvesting is somewhat surprising given that only successfully recovered cells can be used as part of any therapeutic. An integral component to our work is the cell recoverability in all culture platforms we have considered. Though there is substantial literature recognizing that during culture, gentle agitation is desirable, this condition is generally left rather vague

Methods

Results

Conclusion