Development and use of a parallel-plate flow chamber for studying cellular adhesion to solid surfaces T.G. van Kooten Laboratory for Materia Technicu, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, the Netherlands
J. M. Schakenraad Department of Histology and Cell Biology and Centre for Biomedical Technology, University of Groningen, Oostersingel 6911, 9713 EZ Groningen, the Netherlands H.C. Van der Mei and H. J. Busscher* Laboratory for Materia Technica, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, the Netherlands
A parallel-plate flow chamber is developed in order to study cellular adhesion phenomena. An image analysis system is used to observe individual cells exposed to flow in situ and to determine area, perimeter, and shape of these cells as a function of time and shear stress. With this flow system the behavior of human fibroblasts spread on glass is studied when exposed to an increasing laminar flow. The flow system appears to be well-suited for following individual cells during detachment. After 75 to 90 min, at a shear
stress of 350 dynes/cm2, more than 50% of the spread cells are detached from the surface. Cells with higher spreading areas stay longer at the glass surface. Cells round up before detaching. Sometimes the cell body is attached to the substratum through a thin filament during det a c h m e n t . At t h e s c a n n i n g e l e c t r o n microscopy level numerous filopodial extensions are observed. Cell material could only rarely be observed at the light or scanning electron microscopic level on the substratum once a cell was detached.
INTRODUCTION
Cellular adhesion and spreading phenomena have been subject to research for many years. It is now recognized that the process of adhesion and subsequent spreading involves several sequential steps, among which are initiation of cell contact, propagation of adhesion, and spreading.' Analysis of the strength of cellular attachment as a result of these sequential steps can be useful in the development of biomedical devices where an intimate cellbiomaterial contact is needed or, alternatively, must be avoided. Both situations are reflected in the design of antithrombogenic vascular grafts: A firm *To whom correspondence should be addressed. Journal of Biomedical Materials Research, Vol. 26, 725-738 (1992) CCC 0021-9304/92/060725-14$4.00 0 1992 John Wiley & Sons, Inc.
VAN KOOTEN ET AL.
726
attachment is needed when endothelial cells are seeded on the inner wall in order to create a natural lining: whereas without the presence of endothelial cells the inner wall has to be nonadhesive for platelets and erythrocytes? Obviously both approaches set different requirements to the biomaterial surface. In vitro, cell adhesion has been studied in so-called static as well as dynamic systems. In static systems, the number of adhering cells and their morphology are studied in the absence of fluid shear. Schakenraad et al.4found a sigmoidal relation between the area of spread cells and the surface free energy of the substrata in a static system and concluded that substratum surface free energy is a dominant factor in the initial stages of cellular attachment to surfaces. Other factors include proteins adsorbed to a surface before cell contact. Horbett and Schway5 showed an approximately linear correlation between cell spreading and fibronectin adsorption to a series of substrata with increasing wettability (glass and a series of p(HEMA-EMA) copolymers). In dynamic systems, the influence of shear stress on cellular attachment can be studied. The parallel-plate flow chamber has been used predominantly to study the behavior and biochemical responses of endothelial cells in the presence of physiological flow conditions6-' as well as the deposition kinetics of particles and bacteria.' Detachment of cells, and also platelets, has been studied mainly in other devices. Frequently used are the spinning disc"' and the cone-plate device," which have the advantage that a range of shear stresses is simultaneously available in the plane normal to the rotation axis. Because of the rotations, however, the process of detachment cannot be visualized continuously and only cell populations can be followed discontinuously. Recently, Sjollema et al.9 have developed a system in which deposition of microorganisms is followed with continuous, automatic image analysis. The advantages of the system developed by Sjollema et al. are multifold: Deposition of microorganisms can be studied directly in situ, no liquid-air interface passages take place in the flow chamber, rinsing in order to remove so-called loosely bound particles is not necessary, and hydrodynamic conditions are well controlled. We expect that full profit can be taken from these advantages when employing a parallel-plate f low chamber in studying adhesion of eukaryotic cells as well. The aim of this paper is twofold: To describe the development of a parallelplate flow chamber for studying cellular detachment phenomena and to evaluate its practical use in observing human fibroblast detachment from a serum-coated glass surface.
MATERIALS A N D METHODS
Flow system Figure 1 shows a schematic presentation of the flow system. Three funnels (Nalgene) can provide either the cell suspension (C), a protein suspension (C if needed), or fixative (F), respectively. The flow chamber is mounted on a
PARALLEL-PLATE FLOW CHAMBER
727 frame qrabber
P F
C
microscope
ow c h a m b e r
feedback P t l O O
~ _t-_ _
Figure 1. Overview of the complete flow system, including the image analysis components and the heating systems. The damping vessel serves to exclude pulsations in the flow.
microscope stage and placed in a loop consisting of a double-walled vessel, a peristaltic pump (Watson-Marlow 503U with 8-mm inner diameter tubing) and a closed funnel. The compressed air in the funnel dampens the pulsations of the pump to ensure a steady flow. The pressure fluctuations were kept within 1.0% of the mean pressure generated. The pump speed can be adjusted to the required flow rate. Circulating medium is kept at 37°C by the thermostatted double-walled vessel. The parallel-plate flow chamber itself is depicted in Figure 2. The chamber consists of a nickel-coated brass bottom part and a poly(methylmethacry1ate) top part which encloses two plates with measures 7.6 X 5.0 X 0.2 cm (1 X w X h) separated from each other through two spacers. The effective chamber dimensions are 7.6 X 3.8 X 0.02 cm (1 X w X h). The width and height of the inlet and outlet regions gradually change to the chamber dimensions in order to enhance the formation of an established flow. The chamber itself can be heated by four 33R power resistors, mounted on the sides of the bottom part, connected parallel to each other, and regulated by a TOHO TM-48 commander. Feedback is provided by a PtlOO thermocouple, assembled in the top part in the downstream compartment.
Shear stress and laminar flow in a parallel-plate flow chamber
A uniform flow profile can only be present if the flow is steady, incompressible, laminar and establi~hed.’~,’~ The development of such a profile requires a distance Le, which is defined by Eq. (1) for a rectangular cross section: Le = a * h * Re (1)
VAN KOOTEN ET AL.
728
polnl~"
rec, s t c r s
Figure 2. Detailed view of the flow chamber, showing the interchangeable plates, the Pt-100 thermocouple mounted in the downstream compartment, and the O-rings which serve to keep the chamber leakage-free.
in which a is a proportionality constant, h is the chamber height (cm), and R e (Reynolds number) is:
with p the fluid density (g/cm3),which can be taken 1 for tissue culture media, p the viscosity (0.007 g/cm/s at 37"C), w the chamber width (cm), and Q the flow rate (mL/s). Constant u is reported to be between 0.013 (ref. 12) and 0.044.13 In order to conform to a uniform flow profile over the entire plate length, Le has to be small enough compared to the length 1 of the chamber. Generally, flow through straight capillaries of any cross-sectional shape is described by the classical Helmholtz-Smoluchowski equation, often used in electrokinetic measurements. This equation describes a linear relation between streaming potential changes and pressure drops. Deviations from linearity have given rise to the following criterium for Le:
Le
J. M. Schakenraad Department of Histology and Cell Biology and Centre for Biomedical Technology, University of Groningen, Oostersingel 6911, 9713 EZ Groningen, the Netherlands H.C. Van der Mei and H. J. Busscher* Laboratory for Materia Technica, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, the Netherlands
A parallel-plate flow chamber is developed in order to study cellular adhesion phenomena. An image analysis system is used to observe individual cells exposed to flow in situ and to determine area, perimeter, and shape of these cells as a function of time and shear stress. With this flow system the behavior of human fibroblasts spread on glass is studied when exposed to an increasing laminar flow. The flow system appears to be well-suited for following individual cells during detachment. After 75 to 90 min, at a shear
stress of 350 dynes/cm2, more than 50% of the spread cells are detached from the surface. Cells with higher spreading areas stay longer at the glass surface. Cells round up before detaching. Sometimes the cell body is attached to the substratum through a thin filament during det a c h m e n t . At t h e s c a n n i n g e l e c t r o n microscopy level numerous filopodial extensions are observed. Cell material could only rarely be observed at the light or scanning electron microscopic level on the substratum once a cell was detached.
INTRODUCTION
Cellular adhesion and spreading phenomena have been subject to research for many years. It is now recognized that the process of adhesion and subsequent spreading involves several sequential steps, among which are initiation of cell contact, propagation of adhesion, and spreading.' Analysis of the strength of cellular attachment as a result of these sequential steps can be useful in the development of biomedical devices where an intimate cellbiomaterial contact is needed or, alternatively, must be avoided. Both situations are reflected in the design of antithrombogenic vascular grafts: A firm *To whom correspondence should be addressed. Journal of Biomedical Materials Research, Vol. 26, 725-738 (1992) CCC 0021-9304/92/060725-14$4.00 0 1992 John Wiley & Sons, Inc.
VAN KOOTEN ET AL.
726
attachment is needed when endothelial cells are seeded on the inner wall in order to create a natural lining: whereas without the presence of endothelial cells the inner wall has to be nonadhesive for platelets and erythrocytes? Obviously both approaches set different requirements to the biomaterial surface. In vitro, cell adhesion has been studied in so-called static as well as dynamic systems. In static systems, the number of adhering cells and their morphology are studied in the absence of fluid shear. Schakenraad et al.4found a sigmoidal relation between the area of spread cells and the surface free energy of the substrata in a static system and concluded that substratum surface free energy is a dominant factor in the initial stages of cellular attachment to surfaces. Other factors include proteins adsorbed to a surface before cell contact. Horbett and Schway5 showed an approximately linear correlation between cell spreading and fibronectin adsorption to a series of substrata with increasing wettability (glass and a series of p(HEMA-EMA) copolymers). In dynamic systems, the influence of shear stress on cellular attachment can be studied. The parallel-plate flow chamber has been used predominantly to study the behavior and biochemical responses of endothelial cells in the presence of physiological flow conditions6-' as well as the deposition kinetics of particles and bacteria.' Detachment of cells, and also platelets, has been studied mainly in other devices. Frequently used are the spinning disc"' and the cone-plate device," which have the advantage that a range of shear stresses is simultaneously available in the plane normal to the rotation axis. Because of the rotations, however, the process of detachment cannot be visualized continuously and only cell populations can be followed discontinuously. Recently, Sjollema et al.9 have developed a system in which deposition of microorganisms is followed with continuous, automatic image analysis. The advantages of the system developed by Sjollema et al. are multifold: Deposition of microorganisms can be studied directly in situ, no liquid-air interface passages take place in the flow chamber, rinsing in order to remove so-called loosely bound particles is not necessary, and hydrodynamic conditions are well controlled. We expect that full profit can be taken from these advantages when employing a parallel-plate f low chamber in studying adhesion of eukaryotic cells as well. The aim of this paper is twofold: To describe the development of a parallelplate flow chamber for studying cellular detachment phenomena and to evaluate its practical use in observing human fibroblast detachment from a serum-coated glass surface.
MATERIALS A N D METHODS
Flow system Figure 1 shows a schematic presentation of the flow system. Three funnels (Nalgene) can provide either the cell suspension (C), a protein suspension (C if needed), or fixative (F), respectively. The flow chamber is mounted on a
PARALLEL-PLATE FLOW CHAMBER
727 frame qrabber
P F
C
microscope
ow c h a m b e r
feedback P t l O O
~ _t-_ _
Figure 1. Overview of the complete flow system, including the image analysis components and the heating systems. The damping vessel serves to exclude pulsations in the flow.
microscope stage and placed in a loop consisting of a double-walled vessel, a peristaltic pump (Watson-Marlow 503U with 8-mm inner diameter tubing) and a closed funnel. The compressed air in the funnel dampens the pulsations of the pump to ensure a steady flow. The pressure fluctuations were kept within 1.0% of the mean pressure generated. The pump speed can be adjusted to the required flow rate. Circulating medium is kept at 37°C by the thermostatted double-walled vessel. The parallel-plate flow chamber itself is depicted in Figure 2. The chamber consists of a nickel-coated brass bottom part and a poly(methylmethacry1ate) top part which encloses two plates with measures 7.6 X 5.0 X 0.2 cm (1 X w X h) separated from each other through two spacers. The effective chamber dimensions are 7.6 X 3.8 X 0.02 cm (1 X w X h). The width and height of the inlet and outlet regions gradually change to the chamber dimensions in order to enhance the formation of an established flow. The chamber itself can be heated by four 33R power resistors, mounted on the sides of the bottom part, connected parallel to each other, and regulated by a TOHO TM-48 commander. Feedback is provided by a PtlOO thermocouple, assembled in the top part in the downstream compartment.
Shear stress and laminar flow in a parallel-plate flow chamber
A uniform flow profile can only be present if the flow is steady, incompressible, laminar and establi~hed.’~,’~ The development of such a profile requires a distance Le, which is defined by Eq. (1) for a rectangular cross section: Le = a * h * Re (1)
VAN KOOTEN ET AL.
728
polnl~"
rec, s t c r s
Figure 2. Detailed view of the flow chamber, showing the interchangeable plates, the Pt-100 thermocouple mounted in the downstream compartment, and the O-rings which serve to keep the chamber leakage-free.
in which a is a proportionality constant, h is the chamber height (cm), and R e (Reynolds number) is:
with p the fluid density (g/cm3),which can be taken 1 for tissue culture media, p the viscosity (0.007 g/cm/s at 37"C), w the chamber width (cm), and Q the flow rate (mL/s). Constant u is reported to be between 0.013 (ref. 12) and 0.044.13 In order to conform to a uniform flow profile over the entire plate length, Le has to be small enough compared to the length 1 of the chamber. Generally, flow through straight capillaries of any cross-sectional shape is described by the classical Helmholtz-Smoluchowski equation, often used in electrokinetic measurements. This equation describes a linear relation between streaming potential changes and pressure drops. Deviations from linearity have given rise to the following criterium for Le:
Le
