Elsevier

Methods

Volume 56, Issue 3, March 2012, Pages 358-365
Methods

Automated flow cytometry for monitoring CHO cell cultures

https://doi.org/10.1016/j.ymeth.2012.03.001Get rights and content

Abstract

Flow cytometry has been used to accurately monitor cell events that indicate the spatio-temporal state of a bioreactor culture. The introduction of process analytical technology (PAT) has led to process improvements using real-time or semi real-time monitoring systems. Integration of flow cytometry into an automated scheme for improved process monitoring can benefit PAT in bioreactor-based biopharmaceutical productions by establishing optimum process conditions and better quality protocols. Herein, we provide detailed protocols for establishing an automated flow cytometry system that can be used to investigate and monitor cell growth, viability, cell size, and cell cycle data. A method is described for the use of such a system primarily focused on CHO cell culture, although it is foreseen the information gathered from automated flow cytometry can be applied to a variety of cell lines to address both PAT requirements and gain further understanding of complex biological systems.

Introduction

New challenges in quality control of mammalian cell culture bioreactor processes have led to investigations into continuous real-time or near-real-time quality assurance monitoring, with the aim of improvement in process control and compliance [1], [2], [3], [4], [5], [6]. This challenge has given rise to many developments in different monitoring technologies and process analytical technology (PAT) framework has supported the rise of such technologies. The PAT framework was published in a guidance document by the FDA in September 2004 [7] and, subsequently, the introduction of the Quality by Design (QbD) initiative [4], [6] has embraced PAT under its umbrella as a key concept for process management. In biopharmaceutical manufacturing PAT can give an enhanced understanding and control of the production processes.

In situ sensors such as pH, dissolved oxygen, optical density, capacitance, and temperature probes can be used to monitor culture parameters for feedback control of cell culture bioreactors [8], [9], [10], [11]. The ability for tight control of the culture environment improves the reproducibility and scalability of a bioreactor process. Although these parameters help tighten control on the bioprocess it does not give information on viability, cell cycle, cell size, apoptosis, and other parameters that are usually tracked as metrics to indicate reproducibility of the bioprocess [12]. In order to obtain data for some of these parameters off-line analysis needs to occur. This can be accomplished manually, through the use of several analyzers; however, it can be prone to operator error as well as labor increase.

Over the last decade several online and at-line monitoring technologies have been implemented in order to improve data reliability and reduce manual labor involved in cell culture monitoring [13]. Some of these systems include near infrared spectroscopy (NIR) [14], [15], [16], at-line high performance liquid chromatography (HPLC) [3], [17], [18], flow injection analysis (FIA) systems [19], [20], [21], at-line trypan blue exclusion analysis [22] and, to a lesser extent, at-line enzymatic assay systems [12]. Some of these technologies still pose significant challenges to be able to enter a bio-manufacturing environment when it comes to calibration and data analysis, but they can offer significant advantages in establishing a PAT integrated bioprocess environment for improved process monitoring and control [4].

There are systems which use computer imaging techniques, first introduced in the early 90s, for use in suspension cultures [23], able to measure cell morphology, viability, and cell number via the trypan blue exclusion method. These are, essentially, automated at-line microscopes, such as the Cedex HiRes (Roche Applied Science, Innovatis) [24] and Vi-Cell (Beckman Coulter) [25], which can give cell number and viability information through image analysis, although the design of these systems lack the ability to expand their capabilities and obtain information on a greater number of cell physiological parameters (i.e. cell cycle, apoptotic stages, mitochondrial activity, and other organelles which may impact the process) that may be of importance in the bioprocess and cannot be assessed by image analysis alone.

One feature that is required by current at-line systems is an automated sampling component in order to be able to take samples for analysis without introducing contamination into the system. There are several systems on the market that can be used for prevention of bioreactor contamination when sampling although none of them have demonstrated full acceptance in a cGMP environment for the manufacturing of biologics, and this may take time to be established as such a change in the process has to be fully assured to not be detrimental or cause a source of contamination. The current systems that are on the market are the Groton ARS-M unit [26], the BioPROBE unit from BBI-biotech [27], the SEG-FLOW® by Flownamics [28] and the MultiTRACE by Trace Analytics [29]. These systems all claim to be able to sample without introducing contamination to the bioreactor, but further studies will need to be done to determine their feasibility for cGMP use.

The detection of cell-to-cell heterogeneity by rapid and reproducible means is the hallmark of flow cytometric analysis. The importance of this capability in industrial bioprocesses means that this analysis technique is useful to both bioprocess research and development laboratories in reference to analyzing molecular and biochemical events of cell populations.

Flow cytometry has been used in laboratories to analyze cells for many years, thus it would seem fit to use this as a PAT tool for the at-line analysis of cell physiology, in order to understand and predict process kinetics for tighter control and improvement of the biopharmaceutical manufacturing process [6], [30], [31]. This technology has the ability to measure the properties of individual particles by producing a single stream of cells/particles that can be subjected to laser or arc lamp light scattering to allow simultaneous multi-parametric analysis of the physical and/or chemical characteristics of up to thousands of cells/particles per second. Using this principle it is possible to measure cell viability, cell number, cell cycle, apoptosis, mitochondrial activity, and many more cell physiological parameters.

Obtaining cell cycle, cell size, and apoptosis data is very useful in determining the physiological state of the culture, if the culture is reproducible, and the possibility of the culture being utilized for feedback control loops. Cell size has been shown to be a good determinant of productivity [32] while the cell cycle, in particular, has been shown to be an important parameter involved in temperature-induced control between growth and productivity phases of cultures [33], [34] of the bioprocess, which can be an indicator of the appropriate time for nutrient feed introduction [35]. Al-Rubeai et al. showed that the fraction of S/G2 cells were affected by media nutrient composition while monitoring hybridoma cells and these fractions can help predict changes in viable cell numbers following perturbations in culture conditions [36], [37]. In addition, the cell cycle has been used to specifically monitor growth in batch, continuous and perfusion cultures [38] in addition to leading to the possibility for use in the control of perfusion culture feeding rate [39].

Flow cytometer (FC) technology traditionally uses a fluidics system that consists of a central channel/core through which a sample of particles (charged fluid) is injected, which is then enclosed by a pressurized laminar flowing fluid known as sheath fluid. The combined flow is reduced in diameter and the higher pressure of the particle sample creates a pressure differential between the sheath fluid where the greater the differential, the wider the core will become. The sheath flow forces the central particle charged fluid flow front to become parabolic with the greatest velocity at the central core while the outer fluid wall has a velocity near zero, creating a single file of particles, a process known as hydrodynamic focusing. As long as laminar flow is present, the charged fluid and sheath fluid should not mix. Flow cell technology can differ from system to system as illustrated in this protocol communication, where the flow cell utilized has a slightly different design from traditional systems in order to obtain simultaneous electronic volume and optical measurements (with a 100 W UV mercury arc lamp and 488 nm laser utilized as light sources) in the same spatial location. With the cells suspended in an electrolyte-based sheath fluid, large hydrodynamic forces are created by the equilateral triangular geometry focusing the sample stream to the center of the triangular aperture. The aperture consists of a 100× oil immersion micro-objective to collect fluorescence emissions. A cell passing through this aperture will displace its volume by an equivalent amount of electrolyte, resulting in a measured voltage pulse having a height directly proportional to the volume of the cell [40], which can account for even the slightest change in volume hence size.

Automation of flow cytometry has gained attention, due to the possibility of increased knowledge and understanding gained for near real-time data acquisition on cell cultivation under different environmental pressures. The ability of the increased frequency of sampling for a number of assays provides good spatial–temporal coverage of the culture population regarding nucleic acid and protein changes that occur which can be indicators for potential kinetic changes in growth and productivity.

Typical flow cytometry experiments require manual preparation of the samples, which can involve fixation, permeabilization, staining, centrifugation, and incubation steps. For example, the cell cycle traditionally uses a method that often had an alcohol fixation and permeabilization step followed by nucleic acid staining. This method also requires careful timing and sometimes the removal of RNA which could interfere with the staining protocol. These operations can be laborious and possibly requiring several modifications to introduce automation, in order to reduce the manual steps involved making it straightforward to run analysis several times in a set time period without human intervention.

In bioreactor mammalian cell culture it can be important to analyze the time-dependent changes that occur as the culture progresses. For example, alterations in cell cycle distribution can reflect transient growth conditions (Sitton et al., 2008) dependent on micro environmental perturbations induced by stimulating and/or inhibitory elements, such as media nutrients, pH, temperature, inhibitory metabolic byproducts, and dissolved oxygen levels.

Automation of the sampling, staining and analysis process using commercially available equipment can greatly enhance the analysis of bioreactor cultures and contribute towards PAT initiatives in a biopharmaceutical environment [6], alongside the new insights that can be gained in cell physiology.

Our lab has a specific focus on its application to tighter control of bioreactor cell growth and production. Herein that we describe the protocols required to carry out such investigations. This protocol requires the use of an automation platform consisting of both software and hardware elements that when combined implements a series of automated sampling and sample preparation steps for at-line flow cytometric analysis.

Section snippets

Instrumentation and software

  • 1.

    For the purpose of these protocols the use of the FlowCytoPrep™ (FCP) 5000 (MSP Corporation, MN, USA) automated cell preparation instrument was used as a test bed for protocol development and execution. This instrument has been developed for programmable sample withdrawal, washing, fixing, staining, diluting, and sample injection into a flow cytometer. The FCP uses MS Windows XP.

  • 2.

    Cell Lab Quanta SC (CLQSC) Flow Cytometer (Beckman Coulter, Miami, USA), with MS Windows XP.

  • 3.

    WinAutomation software

Reagent setup

Dissolve 250 mg of MSX in 5.55 ml of 18.2 MΩ ddH2O and aliquot into 1 ml vials after 0.2 μm sterile filtration. Use one vial to add 500 μl–500 ml of CD-CHO media. Store the rest of the MSX at −20 °C storage labeled 250 mM MSX stock solution. To make PBS, dissolve 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4 in 18.2 MΩ 0.2 μm filtered ddH2O. Adjust the pH to 7.4 and adjust the volume to 1 l with additional 18.2 MΩ 0.2 μm filtered ddH2O. Sterilize the PBS by autoclaving. Dilute the propidium

Acknowledgments

We thank Christian Lavarreda of MSP Corporation for the use of the FlowCytoPrep 5000 unit. This work was supported by funds from Enterprise Ireland for the apPAT project.

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