Multi-Stream Perfusion Bioreactor Integrated with Outlet Fractionation for Dynamic Cell Culture

Erickson, Patrick; Doshi, Aneesha; Jetley, Gunjan; Amin, Param; Mejevdiwala, Aamena; Patel, Ashna; Bento, Raphaela; Parekkadan, Biju

doi:10.3791/63935

Cited by 2 publications

(3 citation statements)

References 13 publications

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“…These cytokine dynamics suggest that there is a correlation between the introduction of pro-inflammatory TNFα, IL-1β, and IFNγ and the resulting secreted factors by the MSCs in perfusion as well as the PBMCs in the potency assay. Notably, our previous studies found that the mean residence time of the tubing connecting the medium source to the bioreactor was 5.3 h, and the step change should have fully arrived by 6.7 h [31]. The dramatic changes in cytokines occur roughly 6-8 h following the step change, indicating the MSCs likely began to respond immediately to the change in their inflammatory environment.…”

Section: Modeling Inflammation: Viability Potency and Secreted Factor...mentioning

confidence: 96%

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Dynamics of Ex Vivo Mesenchymal Stromal Cell Potency under Continuous Perfusion

Doshi

Erickson

Teryek

et al. 2023

IJMS

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Mesenchymal stromal cells (MSCs) are a candidate for cell immunotherapy due to potent immunomodulatory activity found in their secretome. Though studies on their secreted substances have been reported, the time dynamics of MSC potency remain unclear. Herein, we report on the dynamics of MSC secretome potency in an ex vivo hollow fiber bioreactor using a continuous perfusion cell culture system that fractionated MSC-secreted factors over time. Time-resolved fractions of MSC-conditioned media were evaluated for potency by incubation with activated immune cells. Three studies were designed to characterize MSC potency under: (1) basal conditions, (2) in situ activation, and (3) pre-licensing. Results indicate that the MSC secretome is most potent in suppressing lymphocyte proliferation during the first 24 h and is further stabilized when MSCs are prelicensed with a cocktail of pro-inflammatory cytokines, IFNγ, TNFα, and IL-1β. The evaluation of temporal cell potency using this integrated bioreactor system can be useful in informing strategies to maximize MSC potency, minimize side effects, and allow greater control for the duration of ex vivo administration approaches.

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Section: Modeling Inflammation: Viability Potency and Secreted Factor...mentioning

confidence: 96%

“…Bioreactors were perfused and fractions collected using methods described previously [25,31]. Briefly, culture medium for the perfusion was loaded into syringes and placed in a multi-channel syringe pump (New Era Pump Systems, NE-1600, Farmingdale, NY, USA) kept outside the incubator.…”

Section: Perfusion and Fraction Collectionmentioning

confidence: 99%

Dynamics of Ex Vivo Mesenchymal Stromal Cell Potency under Continuous Perfusion

Doshi

Erickson

Teryek

et al. 2023

IJMS

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“…The general setup and operation of the perfusion system used in this paper are described in detail in our previous publications. [25][26][27] A BioFrac fraction collector (Bio-Rad #7410002) was used to investigate the secretion dynamics over time from HEK293T cells in different conditions. All cell culture secretion time distribution experiments were performed using a multi-channel syringe pump (New Era Pump Systems NE-1600) to act as fresh media (DMEM/F12 -Gibco) reservoirs to be supplied to each bioreactor from 30 mL syringes with BD Luer-Lok Top (VWR).…”

Section: Fraction Collection Set Upmentioning

confidence: 99%

Investigating dynamics of lentiviral vector secretion from HEK293T producer cells using a fractionated perfusion system

Timmins,

Erickson,

Parekkadan

2023

Biotechnology Journal

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Mammalian cell culture is quickly becoming the go to engineering vehicle to mass produce viral vectors in a manner that is safe, convenient, reproducible, and cost and scale effective. Human embryonic kidney (HEK293) cells, in particular, have been utilized and customized (via differentiated transgene expression, modified culture parameters, addition of cytostatic culture agents) to increase vector yields. However, less attention has been made to understanding innate processes within the cells (such as, immune response, cell cycle, metabolism) themselves to better control or increase viral vector product yield. Accordingly, herein, we study the variation in viral production from HEK cells over time using a one‐way perfusion system and bioreactor to study the impact of external factors on secretion dynamics without retrotransduction. Specifically, we study the impact of cell density on viral titer, transduction efficiency, and LDH. Next, we look at the impact of using an inflammatory reporter cell line on viral output, and the secretion dynamics from HEK cells when we use sodium butyrate (cell cycle arrest agent). Lastly, we assess how downregulation of the PDK pathway increases viral titer. Altogether, we investigated the impact of various interventions to increase transient protein expression and viral output from HEK cells in a controlled and measurable environment to ultimately increase the efficiency of HEK cells for downstream clinical applications.This article is protected by copyright. All rights reserved

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Multi-Stream Perfusion Bioreactor Integrated with Outlet Fractionation for Dynamic Cell Culture

Cited by 2 publications

References 13 publications

Dynamics of Ex Vivo Mesenchymal Stromal Cell Potency under Continuous Perfusion

Dynamics of Ex Vivo Mesenchymal Stromal Cell Potency under Continuous Perfusion

Investigating dynamics of lentiviral vector secretion from HEK293T producer cells using a fractionated perfusion system

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