Cell culture on microcarriers is an increasingly important technique for the production of large cell quantities and the scale-up of Cell Therapy applications. Microcarriers—small, spherical beads used to grow adherent cells in a suspension culture—offer several advantages for large-scale cell culture: Because they are suspended, microcarriers allow for a maximized surface area-to-volume ratio. Single-vessel conditions make microcarriers amenable to scale-up applications. Microcarriers also offer the ability to monitor and control physiological conditions when used in bioreactors.
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 will aim to answer all of your questions about dissolvable microcarriers as a next generation technology for stem Cell Therapy.
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The advantages of moving into microcarrier based culture can be realized at relatively small scale. Operation of a bioreactor with a 2.5L working volume using 2 g/L of Corning Dissolvable Microcarriers equates to approximately 25000 cm² of available surface area for cell culture while occupying a small footprint of bench space. The equivalent to this in multi-layer flasks requires either many individual flasks or a few large units that both involve manipulation challenges and labor. A move away from static multi-layer flasks to microcarriers would be ideal, as long as sophisticated monitoring, control, and automation are desired, asthe ability to maintain set points at optimum ranges can impact yield and target product profile. Having these data fits nicely into Quality by Design approaches and generates a significant amount of data for process understanding and control. Additionally, bioreactor culture greatly simplifies sampling of a culture as it can be performed less invasively with reduced aseptic risk. The data generated from a single bioreactor sample are representative of the whole culture as opposed to potential differences between flasks. Regulatory testing requirements may dictate that each vessel have some level of testing (e.g. cell count, metabolite, filter-integrity testing) which is simplified in bioreactor based cultivation. Lastly, labor costs for sampling as well as consumable costs can be reduced, including media per unit of surface area. So the scale that triggers a change to microcarriers will largely depend on how you weigh the benefits of bioreactor technology.
In my research on microcarrier use, I have read about optimization taking a long time. Do you have any advice on streamlining this process and how does is your product in terms of required optimization?
We offer protocols for the implementation of Corning Dissolvable Microcarriers (DMC) as a means to save time as you get started. We also providetechnical support resources for additional guidance.
We typically advise a process be developed in stages: Start by performing studies with your cells and media on microcarriers in ultra-low attachment multi-well plates to assess attachment to the microcarrier. Studies in spinner flask format can provide an early read on attachment and media conditions for agitated culture. For instance, a spinner flask may indicate if serum concentration needs to be lowered for better cell attachment in the dynamic attachment phase and later increased once attachment is sufficient. Spinner flasks also allow for assessment of the effect of nutrient consumption and metabolite production on cell growth and provide insight into subsequent feeding and/or media exchange requirements. In bench top bioreactors, the initial focus should be on the cell attachment phase and optimizing conditions such as agitation and plant densities in microcarrier culture. We recommended that you operate your bioreactor at an agitation rate that simplysuspends the microcarriers, in order to minimize shear and increase agitation. Consider the use of Poloxamer 188 to help mitigate stresses associated with bioreactor culture. From there, monitor cell growth as compared to static conditions and adjust conditions to maximize growth. Many cell types, including hMSCs, can be propagated on DMC in a bead-to-bead fashion allowing for cell expansion by successive additions of DMC and media, which allows cells to naturally migrate to the new surface areas. This method streamlines the cell expansion phase with less manipulation of the culture. Once you have reached your biomass production, the cell harvest protocol can be followed to gently dissolve the DMC for simple and efficient harvest of cells for downstream use.
Prior to investing too much time in bioreactor process development, we recommend working backwards from your desired production volume. . More specifically, when considering your target manufacturing scale, you can estimate an annual production rate. For example, if you find that you must run your process at 1 g of DMC per liter of media vs. 3g/L in order to meet your needs, this will greatly inform how you start the development of your process and possibly reduce false starts. While all bioreactor cell culture requires an investment in development time,timelines can be shortenedwith Corning’s technical support,.
Have you looked at whether the disposable material or the products used to initiate the dissolve effect the expression of surface markers morphology, or pluripotentcy of MSCs
Appropriate surface marker morphology and maintenance of pluripotentcy is certain to be of interest for a user of Corning Dissolvable Microcarriers (DMC) when producing stem cells. Understanding this, we evaluated expected surface markers, post-harvest pluripotentcy, and karyotype of stem cells across a multitude of different conditions: Our studies showed that this is true for both denatured collagen and Synthemax® coatings in serum containing and serum free conditions respectively. These data were generated for spinner flask culture as well as conventional stirred tank bioreactors. With regard to materials or products used to initiate the dissolution of the microcarriers, all of the results referenced were generated after growth and harvest from DMC using the dissolution protocol. One study in particular looked at multiple passages on DMC under two different expansion regimens. The first was bead-to-bead expansion with periodic addition of fresh DMC and media, and the other was a dissolution harvest and reseed approach. This study was performed on Synthemax® coated beads using serum free media and showed that after seven passages the harvested cells from both experimental arms maintained CD73/CD105 at ~95% and CD14/CD45 at <1% while also being able to subsequently differentiate into adipogenic, osteogenic, and chondrogenic cell lineages. Other studies showed similar results but also demonstrated appropriate CD90 and CD34 markers as well as assessment of appropriate karyotype.
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.
Which types of bioreactors have you used the dissolvable microcarriers in? Specifically have you used these in wave type bags?
We are aware of the use of Corning Dissolvable Microcarriers (DMC) in various bioreactor platforms. Both glass and single use spinner flasks have been used extensively for small scale development work. We have seen DMC used in Eppendorf BioBLU (3c, 14c, and 50c), Millipore Sigma Mobius®, PBS Biotech® Vertical-Wheel™, and Sartorius BIOSTAT B systems. In terms of the rocking bag style of bioreactors, successful use of DMC in the Scinus Cell Expansion™ system has been extensively demonstrated.
There are no special media supplements required for using Corning Dissolvable Microcarriers (DMC): The DMC have been used in an array of serum-containing, serum-free, and chemically-defined media. However, there are some considerations to keep in mind. First, cell culture media with a high metal ion content may require harvest condition optimization, as the dissolution harvest requires the use of the chelating agent EDTA. We recommend washing away all cell culture media with DPBS (without calcium and magnesium) prior to initiating dissolution. The extent to which this can be done in certain reactor systems may leave more media behind. While it may not be appropriate for all processes, we have had success harvesting in serum-free media supplemented with additional EDTA at the addition of harvest reagents. Such methods may increase the time it takes for harvest, so one would first need to evaluate tolerance by the cell line of interest. Finally, we recommend considering the use of Poloxamer 188 to help mitigate stresses associated with bioreactor 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.
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.
Cell attachment to Corning Dissolvable Microcarriers is quite efficient: We typically see greater than 90% attachment as determined by the count of free cells in the supernatant. There is a significant range of time required to attach for various processes, however. The characteristics of cell type and/or media used can impact how readily attachment occurs. In examples of cells cultivated in 5L bioreactors, cell attachment can occur within 4 hours given continuous agitation or may require overnight attachment with an intermittent agitation regimen. As seen with other microcarriers, some cells attach better when processing occurs at ½ of the final working volume, low or no serum present at attachment, and with differing agitation conditions.
We have demonstrated successful growth with many cell types that are used in Cell Therapy applications, as well as standard bioprocess production cell lines. The advantages offered by Corning Dissolvable Microcarriers (DMC) are not limited to a particular cell type but are most valuable for specific applications. Due to the fast time to harvest and the elimination of a microcarrier/cell separation step, DMC are idea for any application where the cell is the product or is a precursor to the harvested product. This would include biomass generation as a part of a cell train for large scale applications. DMC are also advantageous for any cell type that does not tolerate extensive shear or Trypsin digestion during harvest.