We recently finished our Ask the Expert discussion, “Enabling Stem Cell Therapy Biomanufacturing using Dissolvable Microcarriers.” During this Ask the Expert session, we covered topics related to the large scale culture of stem cells for cell therapy and the use of dissolvable microcarriers as an enabling technology. Topics included general questions about the use of microcarriers in cell therapy manufacturing, for example, microcarrier optimization, cell aggregation when using microcarriers, and recommended scale for switching to microcarriers. In addition, we had several questions related to the use of dissolvable microcarriers that included, compatible cell types, cell attachement, cell harvest, media, bioreactor compatibility, and durability.
Cell culture on microcarriers is an increasingly important technique for the production of large cell quantities and the scale-up of cell therapy applications. As cell therapies become more common in clinics, there is need for more streamlined scale-up, process robustness, cost efficiency, and regulatory compliance. To meet this need, Corning® has developed a new generation of microcarriers which can be dissolved with a solution of EDTA and pectinase. This new dissolvable microcarrier technology allows cells to be recovered without the need for microcarrier separation. This gentle cell harvest is more ideal than traditional microcarriers for cell therapy applications.
This Ask the Expert session was hosted by Ryann Russell, Sr. Product Development Scientist, Corning Life Sciences. Ryann has over 15 years of experience in the life science sector across a wide range of applications. He studied Biochemistry/Biotechnology and received his BA from Michigan State University. From there he worked as a laboratory assistant at Rockefeller University for 3 years working on bacteriophage interactions with Bacillus species. Subsequently, he began his experience in the bioprocess industry at Merck & Co. where for 12 years he developed upstream processes for vaccine production. In that role he specialized in cell culture and fermentation process development, scale-up, and technology transfer. Ryann joined Corning in 2016 as a Sr. Product Development Scientist / Development Associate where he helps develop novel solutions to problems in the bioprocess industry.
Below is a sneak peek of the discussion, for a full transcript, please see – Ask the Expert – Enabling Stem Cell Therapy Biomanufacturing using Dissolvable Microcarriers
Can you provide an overview of the cell harvest procedure vs. traditional microcarriers?
The harvest procedure is available as a detailed resource document that your local Corning sales representative can provide. Essentially, once cells are ready for harvest, a wash step is performed with DPBS to rinse away culture media. This is often performed by settle-aspirate operation. Since Corning Dissolvable Microcarriers (DMC) are made of polygalacturonic acid (PGA) polymer chains cross-linked via calcium ions, dissolution is achieved with the addition of EDTA (which chelates calcium ions and destabilizes the polymer crosslinking), pectinase (which targets degradation of the PGA polymer), and a standard cell culture protease (which breaks down cell and extracellular matrices). Microcarriers are completely dissolved within 10 to 20 minutes. As compared to a traditional microcarrier harvest, a media wash and exchange into buffer occurs followed by proteolytic dissociation in the presence of mechanical shear via agitator mixing. Harvest may be more difficult, depending on on the degree of cell binding to the carrier (i.e.: charge based interactions, confluence, or the amount of extracellular matrix and adhesion proteins produced) or the extent of microcarrier aggregation. This step may require hold times and shear profiles that can negatively impact subsequent performance of the cells. Once cells have been released, the cells must then be physically separated from the microcarriers. Requiring added steps such as low speed batch or continuous centrifugation, settle aspirate, filtration, or mesh-bag sieving to trap carriers. All add processing time, equipment, consumables, and labor. This method also increases the sterile envelope of the process and, in some cases, also increases contamination risk. The more consumables or equipment in a system can contribute to total particle input of a process. Most importantly, yield losses often occur as a result of additional processing, which can greatly impact the amount of usable final material.
How durable are the dissolvable microcarriers for multi-day culture?
Corning Dissolvable Microcarriers (DMC) are sufficiently durable to support multi-day culture, as we determined after fifteen days of cultivation to reach our biomass target. There was no indication that structure or function was negatively impacted at this time point. In terms of physical durability, it is worth nothing that, if DMCs were to become fractured, generated particulates would be dissolved in the harvest step and thus reduce the risk of particulates downstream. This is an advantage over other types of microcarriers where dissolvability is not feasible. Other considerations for microcarrier cultivation should be kept in mind when selecting a bioreactor platform. Similar to other microcarriers on the market, it is recommended that bioreactors be selected that are compatible for their use. For example, agitation systems should not grind and fracture microcarriers. There are single-use bioreactor configurations designed to avoid milling of microcarriers. Additionally, if long term culture circulation is desired (as is the case for perfusion culture), one can utilize pumps that are compatible with microcarriers. Peristaltic pumps can be used for simple transfers but should be avoided for extended recirculation.
Can you explain about the formation of cell aggregates with microcarriers, have you seen this and do you have any advice on how to limit this?
The formation of multiple microcarrier and cell aggregates is a common phenomenon seen for many, if not all, microcarriers under certain conditions. This is driven by cell adhesion molecules (CAMs) that are typically expressed as a consequence of growth and allow cells to adhere to extracellular matrix or other cells. As such, the extent of multi-bead aggregates relates to the amount of turbulent shear in a mixed environment. Depending on the cell line, increasing the agitation rate 12-24 hours after cell attachment will decrease the likelihood of these aggregates forming; however, the micorenvironment produced by such aggregates may be advantageous depending on your culture needs, like 3D approaches. In that case, agitation can be optimized such that aggregates do not become large enough to form stagnant cores leading to cell necrosis. Live/dead staining and other cell health indicators can be useful to defining optimum aggregate size.