Elsevier

Cytotherapy

Volume 21, Issue 1, January 2019, Pages 76-82
Cytotherapy

Closed loop bioreactor system for the ex vivo expansion of human T cells

https://doi.org/10.1016/j.jcyt.2018.10.009Get rights and content

Abstract

Background aim

Translation of therapeutic cell therapies to clinical-scale products is critical to realizing widespread success. Currently, however, there are limited tools that are accessible at the research level and readily scalable to clinical-scale needs.

Methods

We herein developed and assessed a closed loop bioreactor system in which (i) a highly gas-permeable silicone material was used to fabricate cell culture bags and (ii) dynamic flow was introduced to allow for dissociation of activated T-cell aggregates.

Results

Using this system, we find superior T-cell proliferation compared with conventional bag materials and flasks, especially at later time points. Furthermore, intermittent dynamic flow could easily break apart T-cell clusters.

Conclusions

Our novel closed loop bioreactor system is amenable to enhanced T-cell proliferation and has broader implications for being easily scaled for use in larger need settings.

Introduction

Current advances in clinical T-cell therapies hold great promise toward the near-term eradication of specific cancers [1], [2], [3]. While a continuous stream of new cell therapeutic candidates emerges, there has been less focus on the underlying biomanufacturing methods and tools to expand human T cells at commercial scale. Many benchtop-scale experiments typically still use T-flasks, cell culture bags or small bioreactors, whereas larger-scale processes with increased regulatory constraints have shifted to clinical-scale, closed-loop and automated systems [4], [5], [6]. Although large-scale clinical and industrial-scale process are often robust, they are also encumbered by rigid protocols, whereas smaller-scale tools are highly adaptable to rapidly evolving needs.

A scalable system for the expansion of T cells, like other cell types, must match the nutrient needs of the cells as they grow, especially the transport of gases such as oxygen and carbon dioxide. The transport of gases in cell culture is inherently related to the cell culture container; the most widely used bench-scale culture vessels are T-flasks and cell culture bags. Although T-flasks are commonplace tools in nearly every biological laboratory environment, scaling can become cumbersome when large cell quantities are required [7], [8]. Although large vessels exist, such as multi-layered flasks and cell factories, these systems are all open loop and require manual intervention for media exchanges and cell harvesting. Cell culture bag systems open up the opportunity for a different approach to scaling, ranging from as small as 5 mL up to several liters from commercially available sources, to theoretically even larger volumes for custom designs. Scaling becomes more straightforward because the bag sizes can be easily increased, although physical handling of such systems may become an issue at significantly larger volumes. Furthermore, culture bags are amenable to closed loops systems because they can be easily fitted with ports for sterile access. Presently, culture bags are limited in terms of their material composition; typically polyolefin/ethylene-vinyl acetate (EVA) or fluorinated ethylene propylene (FEP). Although all these materials will allow for gas permeation and cell growth, they are less than ideal due to their reduced gas permeation as compared with filter capped flasks [9]. We herein take advantage of a highly gas-permeable silicone rubber material that has demonstrated great success in the culture and maintenance of cells to fabricate our own custom cell culture bag [9], [10], [11]. Another significant part of bench-level cell expansion is the normalization of cell concentration and media replenishment [12], [13]. This process requires the disaggregation of T-cell clusters to properly enumerate the culture. There is currently no system, let alone a closed loop one, that is able to perform this, except from a manual pipetting process. Numerous commercial devices include culture agitation that aims to promote nutrient diffusion into the media but does not shear aggregates apart. We herein assess a new custom and highly gas-permeable cell culture bag with the ability to be integrated into a closed loop system to facilitate the disaggregation of T-cell clusters.

Section snippets

Cell culture

Media for all cell culture followed the same recipe: RPMI 1640 (Gibco, Thermo Fisher Scientific), 1% penicillin-streptomycin (Gibco, Thermo Fisher Scientific), 1% HEPES (Gibco, Thermo Fisher Scientific), 1% sodium pyruvate (Gibco, Thermo Fisher Scientific) and 10% heat-inactivated fetal bovine serum (FBS; Peak FBS).

Jurkat cells (ATCC) were initially seeded at a density of ∼250 k/mL, counted every other day, and renormalized to a concentration of ∼250 k/mL after each count.

Peripheral blood

Intermittent fluidic culture improves T-cell yields and minimizes cell aggregation

The effects of a continuous flow culture on the soluble CD3/CD28–activated proliferative capacity of T cells were first examined. A clear negative effect was observed with continuous flow on T-cell proliferation resulting in roughly a quarter of the initial cells present by day 9 (Figure 1A). We hypothesized that nutrient deprivation was an issue because stimulated T cells formed large clonal aggregates and, therefore, were considered an intermittent flow strategy to disaggregate T-cell

Discussion

Our work describes the ability of a closed loop bioreactor system to facilitate T-cell aggregate dissociation and improve gas transfer properties, leading to higher cell proliferation by using a silicone-based cell culture bag material. We herein take away two major conclusions from flow conditions: (i) the notion that proper T-cell activation and proliferation require physical interaction between cells and blast formation [15], [16], [17], [18], [19], which continuous flow does not provide and

Acknowledgments

This work was supported in part by the Shriners Hospitals for Children (B.P.) and the National Institutes of Health grants R01EB012521 and T32-EB016652 (M.L.)

Disclosure of interests: B.P. is a founder and equity holder of Sentien Biotechnologies, Inc., and they have licensed patents pertaining to mesenchymal stromal cell therapeutics. The authors declare no other competing financial interests. Supplementary information accompanies this article online. Data and materials are available upon

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