A continuous flow cell culture system for precision cell stimulation and time-resolved profiling of cell secretion
Creators: name: Erickson, Patrick Michael
Publisher: name: No Publisher Supplied
Year: 2023

description: Biological phenomena that take place on the minutes-to-hours timescale can be challenging to measure in vitro. While optical or other electronic tools may be useful for observing certain processes automatically, measurements of secretion and uptake rates of solutes by cells typically must be measured by manually sampling culture medium. Experiments requiring very frequent or overnight manual sampling are impractical, and furthermore, frequent handling of cell cultures can disrupt cell behavior, potentially impacting experimental results. Static cell cultures are also not suitable for experiments exposing cells to solute signals on the minutes-to-hours timescale, as solutes can only be added or removed in a step-change manner via medium changes that also produce step-changes in the other medium components. In vivo, cells are exposed to smoothly varying solute concentration profiles, such as pharmacokinetic (PK) profiles of drugs, that vary independently of other solutes. Perfusion cultures can overcome these problems—the constant flow of fresh medium can smoothly vary the chemical environment of cells and be used to continuously collect effluent. However, microfluidic devices are difficult to use and make, and existing macro-fluidic systems typically use complex sets of custom parts and do not account for the significant effects of solute dispersion on the distortion of solute signals.In this thesis, we present a modular, macrofluidic perfusion system made almost entirely of commercially available parts that can input solute signals to and measure secretion and uptake rates from six cell cultures in parallel. The system is low cost (&lt; $8,000) and the methods are easily learned. Cells may seeded in various vessels such as well plates using static culture techniques before being “plugged in” to the perfusion system. By utilizing residence time distribution (RTD) analysis, the effects of solute dispersion on signals can be predicted with high accuracy. Solute pulses or step changes can be produced and input to cell cultures and are reshaped by the RTDs of the flow system components. The secretion and uptake rates of solutes from cells can be measured by collecting the culture effluent streams using a modified fraction collector. The system can easily be modified to accommodate new experimental needs.We first show the design and characterization of the perfusion system, and how to perform RTD analysis. We then discuss how mixing chambers can be added upstream of cell cultures to create PK profiles in vitro and demonstrate this capability with a model of lymphoma chemotherapy treatment. We then present six applications of the perfusion system, in which the following are measured: cell autonomous circadian rhythms in gene expression; stimulus-response dynamics of an NF-κB-driven gene; transgenic protein secretion rate as a function of AAV viral vector dose; the immunomodulatory potency, exosome secretion, and cell death dynamics of an ex vivo mesenchymal stromal cell therapy; the impacts of chemical treatments on lentiviral vector manufacturing rates, vector potency, and cell death; and the dynamics of planktonic cell release and glucose consumption of 3-D models of bacterial biofilms. Finally, future directions are discussed.