Microfluidic Cell Culture

Young, E.W.K. and Beebe, D.J. (2010) fundamentals of microfluidic cell culture in controlled... 

As consequence of implementation of parallelization in microfluidic cell culture chips, detection of biologically relevant cellular parameters imposes further requirements on the development of the applied detection techniques. Using available motorized microscope stages, time-lapse fluorescence microscopy is a widely applied technique in monitoring cellular responses. Alternatively, fluorescent plate readers facilitate real-time monitoring in highly parallelized systems (readouts for 1,536 well microtiter plate format). 

Implementation of fluidic functions inside a plate reader is however not a straightforward task. For short-term detection, the chip depicted in Fig. lOf [16] in the Chapter Perfusion Based Cell Culture Chips of this book could be amenable for use with a plate reader since its function does not require any external pump. However, for long- term real-time monitoring, this system is not suitable due to the need for a CO2 incubator. An alternative approach could be based on a multichannel pump, suitable for integration with a polymeric microfluidic cell culture chip independent of a CO2 incubator. The pump shown in Fig. 10b [17] in the Chapter Perfusion Based Cell Culture Chips of this book could function as the basis for such an approach. 

The approach to use chip based electroanalytical systems to monitor the dynamics of processes in living cells facilitate the possibility to integrate the detection systems to microfluidic cell culture chips. In virtue of the functional principle of such systems, cells can be cultured on the platform where detection takes place. Hence, the measurements can be conducted in an environment that has been tailor-made for proper adaptation to the requirements of the cultured cells. Furthermore, such miniaturized systems possess the capability to achieve operational automation and facilitate measurements... 

In virtue of the characteristic small dimensions and the applied low flow rates, the flow is laminar in perfusion based microfluidic cell culture chips. Consequently, convection only exists in the direction of the applied flow (x-direction), whereas in the directions perpendicular to the flow (y- and z-direction) only diffusion contributes to mass transfer. This is schematically illustrated in Fig. 2b, depicting a pronounced flow in x-direction. Due to short distances, mass transfer by diffusion is sufficiently effective in providing nutrients and removing metabolic waste during continuous perfusion, eliminating formation of concentration gradients, and hence accumulation of metabolic waste. Furthermore, the small dimensions of microfluidic cell... 

Figure 2. A schematic view of the relative magnitude of mass transfer in x, y and z-direction in a (A) traditional cell culture vessel and (B) microfluidic cell culture chip.

The primary goal in using perfusion based microfluidic cell culture chips is to achieve a high degree of control over the microenvironment that the cultured cells are exposed to, i.e. to create a microenvironment that resembles the one cells are exposed to in vivo. In order to illustrate the difference between traditional culture vessels and perfusion based microfluidic cell culture chips, an effective culture volume (ECV) has been proposed as a descriptive concept [4]. ECV is a combined function of the characteristic mode of mass transfer (convection or diffusion), the magnitude of the mass transfer in all directions (x, y, z) in space (Fig. 2) as well as the extent of protein adsorption to the surfaces in a system. In vivo systems, e.g. tissue as part of an organ, are characterized by a fluid volume that is comparable to the volume of the cells in the tissues as well as by diffusive mass transfer. Based on these features the ECV of in vivo systems is small. 

To facilitate a semi-quantitative comparison of mass transfer in traditional in vitro culture vessels and perfusion based microfluidic cell culture chips, the Peclet number (Eq. 1) has been used [4] ... 

When designing perfusion based microfluidic cell culture chips, the primary areas of concern are the type of flow profile a system generates and an appropriate approach for fluid delivery into the system that facilitates the planned cell based experiments. Upon making a structural design that meets the set requirements, the subsequent step comprises the choice of material and method of fabrication suitable for the chosen material. 

Figure 3. A microscope image of microfluidic cell culture chips having (A) channels (type 1) (Reprinted with permission from Ref [5], copyright 2005 The Royal Society of Chemistry) and (B) chambers (type 2) (Reprinted with permission from Ref [6], copyright 2006 American Chemical Society). A schematic view and finite element simulation of the vertical flow profile (velocity field) of type 1 (C and E) and type 2 (D and F).

Figure 4. (A) An image of a microfluidic cell culture chip having 1 mm height difference between the bottom of the chamber and the channels (type 3) (inset An enlargement of a flow equalizer that generates an even lateral distribution of streamlines). (B) A finite element simulation of the vertical flow profile (velocity field) of the system in (A). (Reprinted with permission from Ref [8], copyright 2008 The Chemical and Biological Microsystems Society.)...

Fluid delivery into a microfluidic cell culture chip has three primary purposes (i) cell seeding, (ii) perfusion with culture medium, and (iii) introduction of cellular effectors during an assay. In the simplest format, a system has one inlet, which is used for all three purposes (Fig. 4a). If the fluidic path that is used for cell seeding is long and has multiple branches such as the flow equalizer in Fig. 4a, premature sedimentation of cells along the fluidic path is easily the consequence. In order to alleviate such a problem, a dedicated inlet for cell seeding is recommended such a system is shown in Fig. 7a [7]. The system has, however, a common inlet for perfusion with culture medium and introduction of cellular effectors. 

The most widely used material for fabrication of microfluidic systems, and hence also microfluidic cell culture chips, is PDMS, which in many systems is utilized in combination with glass. PDMS is fabrication wise well suited for prototyping, whereas it is not in the same degree amenable to mass reproduction. A new emerging trend is, however, fabrication using thermoplastic polymers, such as pol5miethylmethacrylate (PMMA), cyclic olefin copolymer (COC) and polycarbonate (PC), which can be used for... 

The efficient gas permeability of PDMS imposes some restrictions to the usage of the fabricated microfluidic cell culture chips. PDMS allows equilibration of the culture medium with oxygen directly from the ambient, and upon need of additional oxygenation, thin PDMS membranes (ca. 100 pm thick) can be used for controlled oxygenation of the culture medium [6]. However, in an analogous way, due to this efficient gas permeability, the chips release CO2 unless kept in a CO2 incubator, thus changing the pH inside the cell culture chamber. Consequently, PDMS-based chips cannot... 

A variety of techniques have been applied for sterilizing microfluidic cell culture chips and covered by reviews [12, 25] and references therein, such as autoclaving, UV light, oxygen plasma, gamma irradiation, ethylene oxide exposure and perfusion with ethanol, hypochlorite or sodium hydroxide. The applicability of the different techniques primarily depends, aside from what is available in a laboratory, on the type of system and the fabrication material. Autoclaving is an effective method but not suitable for chips fabricated of thermoplastic polymers. The applied temperature and pressure... 

Figure 11. Microfluidic cell culture chips for 3D cell culture experiments featuring perfusion...

P.J. Hung, P.J. Lee, P. Sabounchi, R. Lin and L.P. Lee, Continuous perfusion microfluidic cell culture array for high-throughput cell-based assays. Biotechnology and Bioengineering, 89(1), 1-8 (2005). 

R. Gomez-Sjoberg, A.A. Leyrat, D.M. Phone, C.S. Chen and S.R. Quake, Versatile, fully automated, microfluidic cell culture system. Analytical Chemistry, 79(22), 8557-8563 (2007). 

D- FCCS Three-dimensional microfluidic cell culture system... 

Fig. 1 Perfusion culture of the cells in a gel-free 3D microfluidic cell culture system (3D-nFCCS). (a) Representation of a one-pass perfusion culture system, (b) Optimization of perfusion culture flow rate for maximum cell viability [26]...

Fig. 10 (a) Multichannel 3D microfluidic cell culture system consisting of four chambers with 3D cell constmcts, representing four organs potentially involved in drug toxicity studies, (b) Cells are cultured in a closed-loop with medium being circulated as a blood surrogate [87]... 

Fig. 11 Overview of the possible uses for a multiplexed microfluidic cell culture system. Input can be used for control of the stem cell nice and drug testing. The gradient generator creates a concentration profile of the input factors. The cell culture chamber is a perfusion chamber. Analysis involves monitoring the output of the device, which can include imaging with appropriate biomarkers, analysis using Inline sensors or standard laboratory equipment [74]...

Cell Assays in Microfluidics Cell Culture (2D and 3D) on Chip... 

Cell and tissue culture Cellular microenvironments Microfluidic cell culture Microfluidic platforms Organ fabrication Organ or tissue augmentation... 

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