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Physical Parameters to Implement an Intracellular Organelle Separation using Step-SPLITT Fractionation

Poster · June 2013

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 Physical Parameters to Implement an Intracellular
Organelle Separation using Step-SPLITT
Fractionation
Iván Camilo Navarrete Q1,2, Abelino Vargas Jiménez1,2,
Carolina Ochoa Cabezas1,3, Mauricio Hoyos4, Marcela Camacho1,3.
1
Laboratorio de Biofísica, Centro Internacional de Física, Bogotá, Colombia. 2Departamento de Física, Facultad de Ciencias, Universidad Nacional de Colombia,
Bogotá, Colombia.
3
Departamento de Biología, Facultad de Ciencias, Universidad Nacional de Colombia, Bogotá, Colombia.
4
Laboratoire de Physique et mécanique des milieux hétérogènes, UMR7636 CNRS, Ecole Supérieure de physique et chimie industrielles, ESPCI, 10 rue
Vauquelin

Introduction Results and Discussion

Leishmaniasis is a re-emerging tropical disease caused by parasitic protozoa of the genus
Leishmania. The life cycle of this pathogen includes a mosquito vector and a vertebrate.
After entry into its mammalian host, the parasite is taken up by cells of the immune
system, macrophages, and confined into a low pH intra-cellular compartment known as
the parasitophorous vacuole (PV; Fig. 1a).
We are interested in studying the permeability of this compartment assuming that
transport through the membrane of the PV is important for parasite survival. To make the
PV amenable to electrophysiological recordings this compartment should be isolated and
enriched. Separation of PVs can be achieved by macrophage disruption followed by
centrifugation in a differential density gradient (Cortázar et al. 2006). However, the
gradient alters either the volume of the PV or the surface of its membrane.
Based on the Venturi effect, mechanical rupture of macrophages was achieved.
Needle necks of 26G broke cells but also PVs (Fig. 2a). Therefore this process was
optimized and modeled. Rupture was the result of shear forces, volume and pressure
change (Fig. 1b). Once standardized, the diameter and density of PVs were
determined and the values obtained were intermediate compared to non-infected and
infected macrophages (Fig. 2b). Fluid density was similar to that of water and the
carrier fluid has Newtonian behavior (Fig. 2c).
a. b.

Thus, in this study we modeled the separation of this compartment using Step-SPLITT
separation on gravitational mode. Some physical properties of the PV and carrier fluid
were determined for the calculations. A Step-SPLITT channel was built and its
dimensions and geometry used in the model. Based on these parameters a simulation of
the carrier fluid flow and PV trajectory into the Step-SPLITT channel was performed. The
PVs trajectories during their migration through the channel under gravity forces were also
measured. Finally, volumetric flow rates are calculated in order to improve PV separation.

a. b. c.

Figure 3. a. Theoretical hydrodynamic stability found in the simulation using CDF fluent package. b. Fluorescent
microscopy of the trajectories of infected macrophages labeled with acridine orange inside the S-Splitt channel
built.

A S-Splitt channel was built (Fig. 2d) and its hydrodynamic stability modeled (Fig. 3).
The simulation shows a Poiseuille profile a viscous and incompressible fluid with
laminar flow. This result was tested empirically in the channel built where acridine
orange infected macrophages were injected into culture medium showing laminar
Figure 1. PV labeling. Cells of the macrophage-like cell line J774.A1 were infected with Leishmania amazonensis promastigotes and
flow and no interactions with the channel walls.
after 48 hours loaded with 8 µM of the fluorescent cationic dye Acridine orange. This probe inter-calates within nucleic acids and when
excited at 480 nm emits yellowish-green. It also enters into the PV where is protonated and trapped, due to the low pH, and emits
orange-red. a. Light and fluorescent image of infected macro-phage. Note the yellowish-green of the macrophage nucleic acids in the
nucleus and the orange-red of the large PV. b. Light and fluorescent images of Isolated PVs. Note the yellowish-green emission from
parasite nucleic acids against the orange-red of the PV. c. Rupture system and simulation of the Venturi effect done by FLUENT™ CDF
package. Dc Dre Criteria Exit
Dc >
PV 13.7 10.5 Dre a
Experimental Design
In vitro cultured macrophages were infected with Leishmania amazonensis. Cells were harvested at 48 hours Dc <
16.5 Dre b
post-infection, exposed to osmotic shock (Chakraboty et al. 1994) and mechanically disrupted. Rupture was
standardized based on the Venturi effect trough confronted needles attached to syringes (Fig. 1c). After
mechanical disruption various populations of particles were observed: non-infected macrophages, infected Table 2. Calculated critical diameter of PVs and prediction of exit for a channel as the one shown in the accompanying cartoon.
macrophages, isolated PV and cell debris (Fig. 2a). Cell and PV diameters were measured under light
microscopy in a an inverted microscope (Axio Observer.A1, Zeiss). Images were acquired with the program
Axiovision and diameter calculated. Cell diameter was also measured by impedance in a Coulter counter The trajectories for PVs were simulated. The critical diameter was the criteria chose to
(Beckman; Fig. 2b). Cell and PV densities were determined in a differential density gradient made with Percoll
suggest separation. In a total volumetric flow rate of Q=8ml/h, with inlet and outlet
(Sigma; Data: Fig. 2c) and their isopicnic point calculated against standard beads (Sigma Aldrich). The carrier
fluid properties density and viscosity were measured by pycnometry and viscosimetry respectively (Data: Fig. rates of 0.2 and 0.5 respectively, PV of 16.75 µm in diameter will be driven towards
2c). the b exit while smaller PVs will leave the channel through a as well as infected and
a. Cell rupture b. Cell diameter non macrophages (Fig. 4).

U=
( ρ p −ρf )d 2
g
18η

1
d. S-Splitt channel built  2ηQt  2

dc = 3 
 BLg ( ρ − ρ ) 
 p f 

c. Density

diameter (µm) density (g/cm3) Figure 4. Separation trajectories for 16,45 µm vacuoles from non-infected and infected macrophages. Simulation was carried out
based on the fourth-order Runge-Kutta method (Vargas, 2013) (Ratier et al 2010 ).
PV 13.5 1.062
Conclusion.
vicosity (g/cm.s) density (g/cm3) In this study the density of PV was determined for the first time. The PV disruption
Flui was optimized taken advantage of the Venturi effect and PV separation is theoretically
d 1.3 1.007
possible based on the critical diameter.
References
(1) CORTAZAR TANIA, HERNÁNDEZ JOSELÍN, ECHEVERRY MARIA CLARA, CAMACHO MARCELA (2006): Papel de la Vacuola Parasitófora de
Macrófagos de Ratón Infectados por Leishmania Amazonensis en la Adquisición de Moléculas, En: Biomedica 26, 26-37 p.
(2) PRASANTA CHAKRABORTY, SHEILA STURGILL-KOSZYCKI,  DAVID G. RUSSEL (1994) Phagosomes Isolation and Characterization of
Pathogen-Containing Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110.
(3) VARGAS ABELINO, Modelo Físico Para La Separación de Células en un Canal Step-SPLITT Tesis de Maestría Universidad Nacional de
Colombia.2013.
Figure 2. a. Light microscopy of a sample after rupture. Note non-infected, infected macrophages, free PVs and cell debris. b. Cell (4) RATIER CLAIRE & HOYOS MAURICIO. Acoustic Programming in Step-Split-Flow Lateral-Transport Thin Fractionation. Anal. Chem. 2010, 82,
diameter counted by impendence for non-infected and infected macrophages. c. Mean cell diameter and density measured. Mean fluid 1318-1325.
viscosity and density measured. d. Dimensions and images of the S-Splitt channel built. Agknowledges
Support was given by the Colombian Research Founding agency Colciencias trough the projects ECOS-COLCIENCIAS and 22285693354 and
222851928951, Universidad Nacional de Colombia and Centro Internacional de Fisica.
Corresponding Authors
icnavarreteq@unal.edu.co/mmcamachon@unal.edu.co
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