Dissolvable Gelatin‐Based Microcarriers Generated through Droplet Microfluidics for Expansion and Culture of Mesenchymal Stromal Cells

Abstract

Microcarriers are synthetic particles used in bioreactor‐based cell manufacturing of anchorage‐dependent cells to promote proliferation at efficient physical volumes, mainly by increasing the surface area‐to‐volume ratio. Mesenchymal stromal cells (MSCs) are adherent cells that are used for numerous clinical trials of autologous and allogeneic cell therapy, thus requiring avenues for large‐scale cell production at efficiently low volumes and cost. Here, a dissolvable gelatin‐based microcarrier is developed for MSC expansion. This novel microcarrier shows comparable cell attachment efficiency and proliferation rate when compared to several commercial microcarriers, but with higher harvesting yield due to the direct dissolution of microcarrier particles and thus reduced cell loss at the cell harvesting step. Furthermore, gene expression and in vitro differentiation suggest that MSCs cultured on gelatin microcarriers maintain trilineage differentiation with similar adipogenic differentiation efficiency and higher chondrogenic and osteogenic differentiation efficiency when compared to MSCs cultured on 2D planar polystyrene tissue culture flask; on the contrary, MSCs cultured on conventional microcarriers appear to be bipotent along osteochondral lineages whereby adipogenic differentiation potential is impeded. These results suggest that these gelatin microcarriers are suitable for MSC culture and expansion, and can also potentially be extended for other types of anchorage‐dependent cells.

A Scalable System for Generation of Mesenchymal Stem Cells Derived from Induced Pluripotent Cells Employing Bioreactors and Degradable Microcarriers

“…Recently, Ng et al reported a single‐channel droplet‐fluidics device with the capacity to generate 180 μm diameter genipin‐crosslinked gelatin spheres. 49 The rate of sphere production in the Ng study required 10 hours of run time to supply a 100 mL culture which is 50 times slower than what was achieved with our 100 channel system. Furthermore, genipin crosslinking of gelatin took 2 days to complete compared with seconds for photocrosslinking GelMA.…”

Microcarriers in application for cartilage tissue engineering: Recent progress and challenges

“…The biodegradability of gelatin microcarriers can be achieved by collagenase. For instance, Ng et al fabricated dissolvable gelatin-based microcarriers for MSC expansion [ 176 ]. Another study showed that the gelatin microcarriers could enhance the efficiency of chondrogenesis in bone marrow stromal cells (BMSCs) in vitro [ 177 ].…”

Challenges and opportunities in downstream separation processes for mesenchymal stromal cells cultured in microcarrier‐based stirred suspension bioreactors

“…Methods where the detachment efficiency and cell viability were greater than 90% were considered “Best,” whereas results in the range of 60%−90% were considered “Moderate,” and less than 60% were deemed “Inadequate.” Furthermore, the scalability of the responsive microcarrier detachment process was also assessed as it is significantly different from scaling up traditional enzymatic detachment methods. For methods implemented on a production scale (>1 L), they were considered “Best,” and small‐scale implementation (<1 L) was considered “Inadequate” (Dosta et al, 2020; Hanga et al, 2020; C. Li et al, 2016; Narumi et al, 2020; Ng et al, 2021; Yan et al, 2020; Yang et al, 2010; Yuan et al, 2018).…”

“…In this case, the primary target of the enzymes is the cleavage of microcarriers rather than cellular membrane proteins. Ng et al (2021) expanded hBM‐MSCs on gelatin microcarriers in a spinner flask and obtained harvesting efficiencies of 93% after exposing the microcarriers to pronase for 30 min (Ng et al, 2021). Dosta et al (2020) expanded human MSCs (hMSCs) on gelatin microcarriers in a 3 L bioreactor and dissolved the microcarriers using a combination of a reducing agent called tris(2‐carboxyethyl)phosphine hydrochloride (TCEP) and 0.025% Trypsin, achieving cell viabilities greater than 90% (Dosta et al, 2020).…”