Long term expansion profile of mesenchymal stromal cells at protein nanosheet-stabilised bioemulsions for next generation cell culture microcarriers.

Lihui Peng,Julien E Gautrot

doi:10.1016/j.mtbio.2021.100159

Abstract

Tremendous progress in the identification, isolation and expansion of stem cells has allowed their application in regenerative medicine and tissue engineering, and their use as advanced in vitro models. As a result, stem cell manufacturing increasingly requires scale up, parallelisation and automation. However, solid substrates currently used for the culture of adherent cells are poorly adapted for such applications, owing to their difficult processing from cell products, relatively high costs and their typical reliance on difficult to recycle plastics and microplastics. In this work, we show that bioemulsions formed of microdroplets stabilised by protein nanosheets displaying strong interfacial mechanics are well-suited for the scale up of adherent stem cells such as mesenchymal stromal cells (MSCs). We demonstrate that, over multiple passages (up to passage 10), MSCs retain comparable phenotypes when cultured on such bioemulsions, solid microcarriers (Synthemax II) and classic 2D tissue culture polystyrene. Phenotyping (cell proliferation, morphometry, flow cytometry and differentiation assays) of MSCs cultured for multiple passages on these systems indicate that, although stemness is lost at late passages when cultured on these different substrates, stem cell phenotypes remained comparable between different culture conditions, at any given passage. Hence our study validates the use of bioemulsions for the long term expansion of adherent stem cells and paves the way to the design of novel 3D bioreactors based on microdroplet microcarriers.

Highlights

Since the discovery of mesenchymal stromal cells (MSCs) in bone marrow in the late 1960s [1,2], Mesenchymal stromal cell (MSC) have been isolated from almost every tissue in the human body [3,4]
The selection of protein nanosheets for the stabilisation of bioemulsions supporting MSC adhesion and expansion was based on several key criteria: 1. The formation of mechanically strong protein nanosheets able to resist cell-mediated contractile forces generated during cell spreading and migration; 2
Cell adhesion to liquids has been reported through simple adsorption from medium proteins and direct fibronectin adsorption, we did not select these systems as we found that the former was not supporting MSC adhesion, whereas the latter did not promote the formation of stable emulsions and was reported to be very dependent on oil type

Summary

Introduction

Since the discovery of mesenchymal stromal cells (MSCs) in bone marrow in the late 1960s [1,2], MSCs have been isolated from almost every tissue in the human body [3,4]. MSCs should express CD73, CD90 and CD105 whilst remaining negative for CD45, CD34, CD14 or CD11b, CD79a or CD19 and HLA-DR [5] Because of their self-renewal ability and multi-potency, they are one of the most widely used cell source for clinical applications, whether for cell therapy or tissue engineering [5,6,7,8]. More recent studies revealed the potential of MSCs to treat Covid-19 induced pneumonia [11,12]. In most of these applications, high cell numbers need to be delivered, often in the tens of millions per treatment. Since MSCs are relatively sparse and rather difficult to isolate from patients in large numbers, novel cell manufacturing platforms are required for the scale up and automation of processes

Methods

Results

Conclusion