A continuous flow cell culture system for precision cell stimulation and time-resolved profiling of cell secretion

Erickson, Patrick; Houwayek, Tony; Burr, Alexandra; Teryek, Matthew; Parekkadan, Biju

doi:10.1016/j.ab.2021.114213

Cited by 8 publications

(6 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our continuous-flow perfusion system allowed for precise quantification of the secretion rate of cells over time (Figure 1(b)). 22 PTH secretion was measured over time for five days post-transduction. For each well, PTH secretion averaged about 6 pg per hour at peak expression which occurred around 72 h post-transduction.…”

Section: Resultsmentioning

confidence: 99%

“…A continuous-flow, culture perfusion system optimized in our lab was used as previously described. 22 In brief, a 12-well plate was modified to include plug-flow input and output streams to measure media samples at a rate of 0.2 mL per hour for 120 h. To measure transduction efficiency for all in vitro experiments, GFP-positive cells were counted as a percentage of total cell count using image cytometry on day 3 post-transduction (Celigo, Nexcelom, Lawrence, MA, USA). PTH secretion for HEK293, HepG2, and U2OS cells was determined by transducing cells with the AAV2-EF1a-PTH-IRES-GFP vector.…”

Section: In Vitro Assaysmentioning

confidence: 99%

See 1 more Smart Citation

Bioactive, full-length parathyroid hormone delivered using an adeno-associated viral vector

Burr

Cabahug‐Zuckerman

Castillo

et al. 2022

Exp Biol Med (Maywood)

Self Cite

View full text Add to dashboard Cite

Delivering the parathyroid hormone (PTH) gene has been attempted preclinically in a handful of studies, but delivering full-length PTH (1–84) using adeno-associated viral (AAV) vectors has not. Given the difficulty in achieving therapeutic levels of secreted proteins using gene therapy, this study seeks to determine the feasibility of doing so with PTH. An AAV vector was used to deliver human PTH driven by a strong promoter. We demonstrate the ability to secrete full-length PTH from various cell types in vitro. PTH secretion from hepatocytes was measured over time and a fluorescent marker was used to compare the secretion rate of PTH in various cell types. Potency was measured by the ability of PTH to act on the PTH receptors of osteosarcoma cells and induced proliferation. PTH showed potency in vitro by inducing proliferation in two osteosarcoma cell lines. In vivo, AAV was administered systemically in immunocompromised mice which received xenografts of osteosarcoma cells. Animals that received the highest dose of AAV-PTH had higher liver and plasma concentrations of PTH. All dosing groups achieved measurable plasma concentrations of human PTH that were above the normal range. The high-dose group also had significantly larger tumors compared to control groups on the final day of the study. The tumors also showed dose–dependent differences in morphology. When looking at endocrine signaling and endogenous bone turnover, we observed a significant difference in tibial growth plate width in animals that received the high-dose AAV as well as dose–dependent changes in blood biomarkers related to PTH. This proof-of-concept study shows promise for further exploration of an AAV gene therapy to deliver full-length PTH for hypoparathyroidism. Additional investigation will determine efficacy in a disease model, but data shown establish bioactivity in well-established models of osteosarcoma.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: In Vitro Assaysmentioning

confidence: 99%

Bioactive, full-length parathyroid hormone delivered using an adeno-associated viral vector

Burr

Cabahug‐Zuckerman

Castillo

et al. 2022

Exp Biol Med (Maywood)

Self Cite

View full text Add to dashboard Cite

show abstract

“…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%

“…Despite these studies, there has been limited investigation into the temporal dynamics of MSC potency and how it changes based on the conditions of the microenvironment. To explore the dynamic potency of ex vivo MSCs, we leveraged a continuous perfusion system design that fractionates media in specific time intervals to model the existing therapy and allow us to characterize MSC behavior in greater depth [23,25]. Culture medium perfused through MSC-seeded bioreactors and was collected in outlet fractions at 2 h intervals and were used in subsequent assays to quantify their potency using a T cell stimulation bioassay and a cytokine analysis.…”

Section: Introductionmentioning

confidence: 99%

Dynamics of Ex Vivo Mesenchymal Stromal Cell Potency under Continuous Perfusion

Doshi

Erickson

Teryek

et al. 2023

IJMS

Self Cite

View full text Add to dashboard Cite

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.

show abstract

“…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

Self Cite

View full text Add to dashboard Cite

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

show abstract

A continuous flow cell culture system for precision cell stimulation and time-resolved profiling of cell secretion

Cited by 8 publications

References 23 publications

Bioactive, full-length parathyroid hormone delivered using an adeno-associated viral vector

Bioactive, full-length parathyroid hormone delivered using an adeno-associated viral vector

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

Contact Info

Product

Resources

About