A Microfluidic-Based Microscopy Platform for Continuous Interrogation of Trypanosoma brucei during Environmental Perturbation

Abstract

The African trypanosome, Trypanosoma brucei, is the causative agent of human African trypanosomiasis (HAT). African trypanosomes are extracellular parasites that possess a single flagellum that imparts a high degree of motility to the microorganisms. In addition, African trypanosomes show significant metabolic and structural adaptation to environmental conditions. Analysis of the ways that environmental cues affect these organisms generally requires rapid perfusion experiments in combination with single-cell imaging, which are difficult to apply under conditions of rapid motion. Microfluidic devices have been used previously as a strategy for trapping small motile cells in a variety of organisms, including trypanosomes; however, in the past, such devices required individual fabrication in a cleanroom, limiting their application. Here we demonstrate that a commercial microfluidic device, typically used for bacterial trapping, can trap bloodstream and procyclic form trypanosomes, allowing for rapid buffer exchange via perfusion. As a result, time-lapse single-cell microscopy images of these highly motile parasites were acquired during environmental variations. Using these devices, we have been able to perform and analyze perfusion-based single-cell tracking experiments of the responses of the parasite to changes in glucose availability, which is a major step in resolving the mechanisms of adaptation of kinetoplasts to their individual biological niches; we demonstrate utility of this tool for making measurements of procyclic form trypanosome intracellular glucose levels as a function of changes in extracellular glucose concentrations. These experiments demonstrate that cytosolic glucose equilibrates with external conditions as fast as, or faster than, the rate of solution exchange in the instrument.

Spatial confinement of Trypanosoma brucei in microfluidic traps provides a new tool to study free swimming parasites

“…(C) To explore potential passive and active trap invasion, we chemically fixed T. brucei parasites to investigate their distribution after influx into the chip both as a single influx, and upon double influx. While on average, 75% of traps were invaded by untreated 2913 parasites, only 16.2% of traps had parasites present if the here is complementary to various other valuable methods that have been generated over the last decade, to study various aspects of T. brucei behaviour [13,15,18,19,[30][31][32][33] reviewed in [12]. We divide these methods into 6 groups for further discussion: a) temperature-based, b) chemical-adhesion-based, c) gel-based, d) optical traps; e) droplet-based and f) microfluidics and nanopatterning.…”

“…Equally it does not allow the study of parasite interactions with geometrical structures potentially mimicking anatomical features. Another device generated by Voyton et al [15] is also capable of multiplexing and large scale-screening, and enables perfusion to study parasite biochemistry. This design faces the same limitations as those described for the previously described microfluidic platform.…”

“…Microfluidic devices developed for the study of parasites have been described in several articles. Main methods have been reviewed by Muthinja et al ([12]), and include elastic substrates which allow for the measurement of force transmission during cell migration [13]; nano-and micro-patterns, which can serve as binding sites for motile parasites [14]; micro-pillar arrays that allowing for parasite navigation studies [13]; micro-channels, which have aimed at confining parasites for longitudinal visualization [15]; and organs-on-chip, which have aimed at the replication of in vivo parameters for the study of host-parasite interactions (reviewed in [16,17]). A recent device incorporated encapsulation and cultivation of single parasites in emulsion droplets to investigate parasite growth patterns and parasite population heterogeneity [18,19].…”

Low-Cost Microfluidic Systems for Detection of Neglected Tropical Diseases

“…Voyton et al (68) described the use of a microfluidic system (CellASIC ONIX2) to monitor living trypanosomes. The authors reported a change in the intracellular glucose in Trypanosoma brucei cells when the extracellular glucose concentration was altered.…”

Revealing spatio-temporal dynamics with long-term trypanosomatid live-cell imaging

“…Trypanosoma brucei is an obligate extracellular parasite with an attached flagellum whose beat causes significant distortion of the entire cell body, which complicates live cell imaging [7,8]. Several approaches have been used for short-term imaging of live T. brucei cells, but these methods are not compatible with parasite division, producing apoptotic cells after less than 4 h [9][10][11]. For long-term live-cell imaging, it is possible to gently immobilize the parasite on the surface of an agarose pad [12,13].…”