Leading solutions in culturing, donor screening, and cryopreservation for allogeneic stem cell therapies
Steve Oh, Director of A*STAR’S Stem Cell Bioprocessing Group unveiled innovations in the manufacturing of allogeneic cell therapies at this year's Cell & Gene Therapy Manufacturing & Commercialization Asia Digital Week. Silvia Hnatova revisits the highlights.
ASTEM institute provides solutions for allogeneic manufacturing, including solutions to culturing, donor screening, and cryopreservation.
In the first part of his talk, Dr. Oh explained the screening process of donors who provide mesenchymal stem cells (MSCs).
Several QC steps are implemented to determine the cells with the best potency, followed by validation of the cultures in vitro and in vivo, enabling therapeutic use (protocol modified from Haccok et al., 2017).
Dr. Oh highlighted the current challenges of MSC harvesting, which is limited by the MSC variability linked to donor age.
Donors around 20 years of age have MSCs with a lower and more consistent doubling time, in contrast to the older donors.
Not all donor-derived MSCs are equal and there is a donor-specific variation in cell doubling time and the genetic variation contributes to MSC quality.
To address the issue of MSC variability, Dr. Oh’s group performed a genomic screen of markers of MSC performance from the donors. GSTT1 was identified as a modifier of MSC numbers, with GSTT1-null MSCs having better scalability.
Dr. Oh pointed out that GSTT1 is a DNA marker of MSC scalability and can be used for donor selection before surgical harvest. This could lead to cost reduction when selecting the best MSCs for allogeneic cell therapies.
The next part of Dr Oh’s talk was focused on the pivotal role of serum-free media in the manufacturing of GMP grade MSCs for allogeneic cell therapies.
Dr. Oh stressed that serum-free media are superior due to the variability in sera composition and the risk of viral contamination in serum-based media. His group managed to develop three types of serum-free media, that were demonstrated to promote the proliferative capacity of MSCs.
When using serum-free media, the potency of MSCs was maintained for a longer type in the cultures and the differentiation capacity of MSCs was improved or equivalent compared to DMEM-FBS media.
It was presented that a xeno-free media formulation achieved better performance and better consistency of MSC propagation compared to FBS and hPL medium. In an important comparison to FBS-based media, the MSC expansion rate also improved.
Dr. Oh finished his presentation on serum-free media concluding that ASTEM media can also be applied to large-scale MSC production in bioreactors.
Switching gears, Dr. Oh presented data from the X-Therma collaboration with ASTEM, that aimed to find long-term cell storage solutions.
Dr. Oh introduced XT-Thrive® cryopreservant that is DMSO-free, chemically defined, and there is a GMP-grade version currently in development.
Eradicating DMSO from cryopreservants is key to reducing toxicity among patient-derived MSCs, Dr. Oh stressed. Dr. Oh’s group evaluated XT-Thrive® on patient bone marrow-defined MSCs to evaluate their properties after thawing.
Data from an experiment in which cultured MSCs were grown in monolayers in serum-containing media and serum-free media, were presented (pre-freeze incubation).
XT-Thrive® demonstrated the improvements to viability up to 24hrs at room temperature compared to CS10 media, which led to a drop in viability of over 30%. This could allow more time to freeze cells, without adding time pressure, Dr. Oh pointed out.
Dr. Oh showed that XT-Thrive® outperformed CD10 in freezing MSCs cultures as monolayers.
This experiment was conducted with cells frozen for one week in liquid nitrogen, expanded in serum-based or serum-free media.
After the thawing, cells were kept at ambient temperature in a cryoprotectant for up to 6 hrs while measuring viability. There was high viability with a minimal drop-off during the ambient temperature processing window.
Moreover, XT-Thrive® led to equivalent recovery after seven days as CS10 in microcarrier suspension culture.
XT-Thrive® led to a higher growth rate than CS10 at 6 days post-thawing. To compare that stable remain unchanged, MSC surface markers were analyzed during culture, post-thaw, and after recovery by Dr. Oh’s groups.
There was no significant difference in gene expression of surface markers, demonstrating that XT-Thrive® does not alter cell identity.
Similarly, colony-forming efficiency was stable and not affected by XT-Thrive®, pointing at the equivalent potency of cells in XT-Thrive® compared to serum-based media.
Dr. Oh concluded that XT-Thrive® use could present a huge advantage in the manufacturing processes, especially in filling and transportation.
