Expansion strategies for human mesenchymal stromal cells culture under xeno‐free conditions

Abstract

Choosing the culture system and culture medium used to produce cells are key steps toward a safe, scalable, and cost-effective expansion bioprocess for cell therapy purposes. The use of AB human serum (AB HS) as an alternative xeno-free supplement for mesenchymal stromal cells (MSC) cultivation has increasingly gained relevance due to safety and efficiency aspects. Here we have evaluated different scalable culture systems to produce a meaningful number of umbilical cord matrix-derived MSC (UCM MSC) using AB HS for culture medium supplementation during expansion and cryopreservation to enable a xeno-free bioprocess. UCM MSC were cultured in a scalable planar (compact 10-layer flasks and roller bottles) and 3-D microcarrier-based culture systems (spinner flasks and stirred tank bioreactor). Ten layer flasks and roller bottles enabled the production of 2.6 ± 0.6 × 10 and 1.4 ± 0.3 × 10 cells/cm . UCM MSC-based microcarrier expansion in the stirred conditions has enabled the production of higher cell densities (5.5-23.0 × 10 cells/cm ) when compared to planar systems. Nevertheless, due to the moderate harvesting efficiency attained, (80% for spinner flasks and 46.6% for bioreactor) the total cell number recovered was lower than expected. Cells maintained the functional properties after expansion in all the culture systems evaluated. The cryopreservation of cells (using AB HS) was also successfully carried out. Establishing scalable xeno-free expansion processes represents an important step toward a GMP compliant large-scale production platform for MSC-based clinical applications. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1358-1367, 2017.

Microcarriers for Upscaling Cultured Meat Production

“…However, the seeding density as well as initial MC concentration should be carefully assessed, as cell growth can be negatively affected due to particle collisions (109) and nutrient concentration gradients cause by diffusion limitations (110). Implementing intermittent stirring has also been reported as an efficient strategy and has been widely used for the expansion of stem cells (111)(112)(113)(114). Lastly, the bioreactor should also be designed in such a way that shear stress is limited and mixing maximized.…”

Aging Donor-Derived Human Mesenchymal Stem Cells Exhibit Reduced Reactive Oxygen Species Loads and Increased Differentiation Potential Following Serial Expansion on a PEG-PCL Copolymer Substrate

“…Maintaining bioreactor systems has the complexity of interconnected bioreactor components and unique contamination risks (e.g., chemical or biological) that complicate the scaling up of this process [ 20 ]. Several groups have noted that optimization of bioreactor yields requires improvement despite the careful monitoring of growth conditions [ 21 ], and that precursor hMSC populations from the original in vivo aspirate are modified and/or lost after 20 population doublings [ 22 ]. Additionally, controlling the size of the hMSC aggregates during a scale-up of the bioreactor risks the aggregates developing necrotic cores that could result in negative consequences for the recipient tissues receiving the cells [ 23 , 24 , 25 ].…”

Successful Use of Human AB Serum to Support the Expansion of Adipose Tissue-Derived Mesenchymal Stem/Stromal Cell in a Microcarrier-Based Platform

“…This prompted us to develop the present study in order to improve the culture conditions to maximize AT MSC production in this bioreactor culture system. Based on our previous reports regarding AB HS use as culture medium supplement for MSC expansion, with cells showing similar doubling times and population doublings compared to cell cultivated with 10% (v/v) FBS supplemented culture medium (dos Santos et al, 2017;Tozetti et al, 2017), we selected this culture medium to perform this study. Thus, aiming to firstly improve the cell adhesion, three different microcarrier coatings were tested in spinner flasks: 10% (v/v) and 20% (v/v) of AB HS and fibronectin.…”