Cell-Expansion Benefits Using Hollow-Fiber Bioreactor Technology

In a November 2022 webinar, Stuart Gibb (scientific strategy lead from Terumo Blood and Cell Technologies) explained the benefits of hollow-fiber bioreactors (HFBRs) for cell therapy. They are suitable for both adherent and suspension-adapted cells. Terumo’s bioreactors are designed to enable flexible feeding and waste-management strategies, which are key features that can facilitate optimal cell growth.

The Presentation
Gibb presented Terumo’s Quantum Flex HFBR platform, which the company launched in September 2022. Bioreactors for the Quantum Flex platform come in two sizes: the standard bioreactor with 2.1 m² of surface area and the small new bioreactor with a surface area of 0.2 m². Each is functionally closed, with material loaded and unloaded using sterile welding.

The standard HFBR contains ~11,500 semipermeable hollow fibers. Each fiber has a diameter ~200 µm, which is relatively large compared with the average chimeric antigen receptor (CAR) T cell, which ranges between
8 µm and 12 µm.

Terumo’s HFBRs have two fluid circuits. Cells introduced to the reactor are cultured along an intracapillary circuit (IC) running within the hollow fibers, whereas the extracapillary circuit (EC) runs outside of the hollow fibers, serving a primary role in gas exchange. Because the fibers are semipermeable, media components, supplements, and other materials can pass between the circuits along a concentration gradient. The HFBR system can operate the IC and EC independently, enabling flexible feeding strategies.

For instance, users can apply the same media formulation in both circuits or program the IC and EC to deliver a cytokine-supplemented medium and a basal medium, respectively. Feeding can be constant or periodic and in either circuit or both. For adherent cells, protocols feed the cells unidirectionally. For suspended cells, users can change the flow through the IC and use bidirectional circulation to keep the cells in the HFBR.

The semipermeable Quantum Flex fibers have a molecular-weight cutoff (MWCO) of about 17 kDa. Items smaller than 17 kDa flow into or out of the fibers along a concentration gradient. For cases in which cytokines weigh
<17 kDa, users may wish to feed both the IC and EC sides of the HFBR.

Waste items such as lactate can pass along a concentration gradient into the EC circuit, which users can direct into the waste bag.

Case Studies
In one study, Gibb’s team used CD3+ T cells that were immunomagnetically isolated, cryopreserved, thawed, activated, and then loaded onto a HFBR system. The expansion took seven to eight days.

For feeding, Gibb’s team used media with human serum and interleukin 2 (IL-2) for the IC side and base media for the EC side. Using material from three different donors, expansion over seven to eight days generated between 13.1 billion and 24.7 billion CD3+ T cells with >96% viability.

Because the T-cell phenotype is critically important to cell-therapy products, Gibb’s team evaluated the phenotypic stability of the cultured cells. The scientists analyzed CD3+
T cells before and after expansion, and then immediately and 24 hours after thawing. The CD4 and CD8 subsets were stable throughout expansion. It also was determined that the stem-cell memory phenotype was enriched throughout the process.

Gibb added that the expansion process did not result in significant terminal exhaustion. During expansion, Gibb’s team analyzed cells for PD-1 and Tim3 markers. Detection of those markers increased only slightly during expansion, ranging 1.7–11% for the different donors.

Questions and Answers
How do you remove cells from a Quantum Flex HFBR? The system can run a harvesting procedure that opens a valve and puts your suspension-adapted cells into a harvest bag. For adherent cells, you will also need a dissociation reagent.

What percentage of cells are recovered from the hollow fibers during harvest? We expect to recover 90–95% of cells from the hollow fibers. We use lactate as a proxy for measurement of cell numbers before harvest, because we can’t directly view the cells when they’re inside the hollow fibers.

Has the Quantum Flex system been used for cell types other than T cells? Many different cell types have been cultured on the platform. It is agnostic to cell types, and we can help you tailor the system to your parameters, whether you’re using adherent or suspension-adapted cells.

Find the full webinar online at www.bioprocessintl.com/category/webinars.