- Email: [email protected]
Microfluidic modulus for convenient cell culture and screening experiments
Microelectronic Engineering 84 (2007) 1694–1697 www.elsevier.com/locate/mee
Microfluidic modulus for convenient cell culture and screening experiments C. Crozatier b
a,*
, I. Tapsoba a, L.P. Xu b, D. Han c, L. Sensebe´ d, Y. Chen
a
a Ecole Normale Supe´rieure, CNRS-UMR 8640, Paris 75005, France Centre for Microfluidic and Nanotechnology, Peking University, 100871 Beijing, China c National Centre for Nanoscience and Technology, 100080 Beijing, China d Etablissement Franc¸ais du Sang Centre-Atlantique, Tours 37020, France
Available online 15 February 2007
Abstract We report on a microfluidic set-up for convenient biological experiments involving cell culture. The strategy is to use a patterned microfluidic cover plate, which can be reversibly bonded to a cell culture substrate, to achieve both long-term cell culture in parallel chambers and control of culture parameters in each chamber. The design is furthermore optimized to enable intrinsic flow rate control. Preliminary results indicate that this novel cells-on-chips concept may lead to the development of microfluidic screening experiments and bio-analysis based on more conventional and high performance observations. 2007 Elsevier B.V. All rights reserved. Keywords: Cell culture; Microfluidics; Screening; Reversible assembly
1. Introduction Microfluidics is a new scientific discipline which is particularly relevant for biological experiments. This is because of the fact that most of the traditional biological experiments are time consuming and laborious. It is highly desirable to develop integrated systems that allow high throughput screening and/or automatic processing of a sequential experimental steps. The concept of ‘‘lab on a chip’’ microfluidic devices fully fits these requirements. In addition, it provides great advantages in the economy of chemical reagents and biological samples. In fact, on-chip cell culture and differentiation studies [1] as well as high throughput cell culture assays [2] have been developed, but they have yet to be compiled and adapted to the precise needs of cell biologists. The main difficulties for such exploration are the fluid control over a long period of time and the fragility of manipulated objects, where slight changes in the parameters can lead to death of all the sam*
Corresponding author. Tel.: +33 1 44322421; fax: +33 1 44322402. E-mail address: [email protected] (C. Crozatier).
0167-9317/$ - see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.mee.2007.01.265
ples. Most of the microfluidic chips developed so far were based on irreversible bonding and hydrostatic pressure or mechanic pumping for fluid control [3]. With these types of cell chips and optimised protocols, it is possible to put a cell population in one stable culture or differentiation condition. However, two issues arise for advanced studies: how to develop screening experiments with parallel and precise fluid control with numerous entries and how to adapt the common used bio-analytical systems to the ‘‘lab on a chip’’ applications. We hereby address these issues by proposing a novel microfluidic modulus that enables inherent flow control through its design and that conveniently gives access to the biological samples after experiments. 2. Experimental 2.1. PDMS device fabrication Soft lithography has been used to fabricate microfluidic devices. With a high resolution (3600 pixel per square inch) laser printer, a transparent plastic film was patterned as
C. Crozatier et al. / Microelectronic Engineering 84 (2007) 1694–1697
mask for contact printing optical lithography. The designed fluidic channels and the patterns for the stamps were then replicated into a 30 lm layer of AZ-9260 photo-resist deposited on a silicon substrate by UV light exposure. Before casting, the resist patterns were exposed to trimethylchlorosilane (TMCS) vapour for 1 min to aid subsequent release. Then, the liquid pre-polymer PDMS was poured on and cured at 80 C for 1 h. After peeling off the cross-linked PDMS structures, if necessary, holes were punched through the elastomer to allow later fluidic access to the reservoirs. The microfluidic device is then washed with ethanol and dried under filtered air flow. No specific treatments were done unless stated otherwise. 2.2. Reversible sealing of PDMS devices with microaspiration A new method has been used to make functional microfluidic channels that can be peeled off from the cell culture substrate [4]. Briefly, in order to realize reversible sealing between the microfluidic channels and the substrate, a network of crossing channels of air has been designed to surround the channels (see Fig. 1). This results in a cavity where vacuum of around 150 mbar can be applied with the help of a mini pump. Such aspiration gives enough strength to hold the microfluidic structure layer and the substrate together with a good sealing, hence allowing various manipulations on the device such as changes of outlets or reservoirs. In addition, the fact that PDMS is permeable to air enables the sucking out of air bubbles trapped in the microfluidic channels. This leads to an easy filling and the certainty that each device is ready for cell culture. Stopping the aspiration will enable the disassembly of the microfluidic device from the substrate. 2.3. Cell culture Rat mesenchymal stem cells were used for cell culture. All details about culture medium and traditional culture
Fig. 1. A schematic figure showing the microfluidic (A) and vacuum (B) channels. The vacuum channels (B) are interconnected, and when vacuum is applied (lower figure), the overall PDMS structure is deformed clamped onto the substrate.
1695
protocols are described elsewhere [4]. Here, the substrate for cell culture is a glass slide coated with Fibronectin (Sigma), a cell adhesion promoter protein, at 4 C over night. Once the glass slide is rinsed and dried, the microfluidic modulus is applied directly onto it. This step must be done in a dust-free environment to prevent leakage. The device is then filled with culture medium by applying droplets of medium at each inlet and outlet and by producing vacuum in the network of crossing channels. The air in the microchannels are then evacuated through the channel walls. Once the cells are loaded into the chambers, a static period of a few hours is required for them to start adhering to the substrate. Afterward, a flow of fresh culture medium is applied at around 100 nL/min to enable cell proliferation.
3. Results and discussion 3.1. Long-term stable flow A stable flow of culture medium in a chamber has proven to be crucial for reliable cell culture protocol. Cells are delicate and complex objects that evolve, quite often unpredictably, with the slightest change in their environment. The shear stress suffered by the cells is quite high because of the dimensions of the chambers (30 lm high). We have conveniently included in the design of the chip an inherent flow control part consisting of a series of channels with outlets in between. Applying an outlet pressure at a different point will result in variation of the overall hydrodynamic resistivity. Two advantages can be taken from this set up. First, we have added overall resistivity to the microfluidic device, hence permitting both better reduction of the variability in the flow rate caused by inherent differences between chambers, and the use of a more stable and convenient flow control device for long-term experiments. In addition, we are now able to regulate the flow rate as we see fit only by choosing the device outlet. This circumvents drastic changes in the predicted flow rate caused by fabrication errors since these unpredictable factors can now be taken into account and the flow rate adjusted in consequence. A vacuum pump has been chosen as a flow control device since it is convenient and stable through time. A vacuum of 300 mbar has been applied at the outlet in order to have a stable underpressure throughout the whole experiment. Too little vacuum is very difficult to stabilize because any slight variation, in the reservoirs’ volume for example, can dramatically change the applied difference of pressure. Here, the overall resistivity of the device was tuned with numerous outlets put into series at the end of the initial one. Each outlet is separated by a microchannel of 100 lm wide and 1 cm long and each is linked to a clamped tube when unused. Fig. 2 shows the variation of the flow rate resulting from the change of outlet: a stable flow with a flow rate as slow as 3 nL/s (corresponding to the 100 nL/
1696
C. Crozatier et al. / Microelectronic Engineering 84 (2007) 1694–1697
Fig. 3. Microphotographs of cell culture in microfluidic chambers: left: cell attachment after seeding; right: cell proliferation after ten days of medium flow.
Fig. 2. Average flow rate variation due to the variation of the overall resistivity of the microfluidic device. Between each output, a channel of 30 lm · 100 lm · 1 cm was added to increase the overall resistivity (see above).
min rate used in our usual on chip cell culture experiments) has been achieved. 3.2. Long-term cell culture On chip cell culture experiments have been described in numerous configurations, but the use of micro-aspired devices is unprecedented. As described in the experimental section, the micro-aspiration technique enables the reversible bonding of the device to the substrate, hence facilitating the retrieval of biological samples once the screening has been done. Moreover, the micro-aspiration technique has proven to be convenient of use for bubble suppression as well. Indeed, screening experiments are cumbersome especially for differentiation parameter screening because of the quantity of conditions to be tested. This has also an impact in microfluidic devices since the number of inlets and the risk of bubble formation increase: a simple bubble can alter the functioning of the whole device. Because a depression is applied all around the microfluidic channels and because the material of the device is made of, PDMS, it is permeable to gas, incomplete filling and bubble formation are no longer of concern. With these advantages in the micro-aspiration technique, it was possible to realize long-term cell culture in parallel chambers. Fig. 3 shows a usual result of an on-chip cell culture experiment where cell proliferation can be observed. Fig. 4 (left) shows one of the microfluidic devices used for parallel cell culture experiments. Each culture chamber was linked to a reservoir that could be filled at will with different substances. Fig. 4 (right) shows a microphotograph of cells in a chamber.
Fig. 4. Left: photography of the long-term cell culture parameters screening device. Right: a microphotography of cells cultivated in a microchamber.
3.3. General applications This device has initially been developed for screening the parameters in the differentiation pathway from MSC stem cells to neural cells [5]. Fig. 5 shows an example of such a differentiation pathway, already tested in one of the
Fig. 5. Left: testing cell differentiation protocol: after each incubation period, the surrounding medium is replaced with other transcription factors. The timing of these changes in the medium is crucial. Here, both SHH and FGF8 factors will vary in concentration. These proteins are crucial for inducing cells into specific differentiation pathways. Right: Immunocytochemistry of rMSC cells engaged in neural differentiation pathway (red: expression of Nestin, a neural protein not expressed in usual rMSC cells; green: fluorescent dyeing for cells; bottom: superposition of both fluorescent image to show that some cells express typical neural proteins, see yellow-colored cells). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
C. Crozatier et al. / Microelectronic Engineering 84 (2007) 1694–1697
authors’ laboratories. With the possibility to add pumps, valves and mixers into a micro-aspired chip, it is now possible to realize a more sophisticated microfluidic modulus that can become an essential tool for screening experiments. Moreover, as there is no restriction for the substrate used with the microfluidic modulus, as long as it is smooth and planar across the aspiration network, we can couple this experiment with analytical devices such as protein or DNA chips for in situ analysis. 4. Conclusion We have successfully applied a novel strategy for onchip microfluidic techniques integration in biological experiments. Cell loading and attachment could be achieved using a substrate that can be subsequently treated and analyzed with traditional methods. Except for the microfluidic device itself, manipulations require only basic materials that can be found in common biological laboratories (tubes, syringes, microscope and vacuum pump). These screening experiments can therefore be performed in a standard cell biology environment. We believe that this
1697
strategy is relevant to achieve a better efficiency in various biological screening experiments. Acknowledgements This work was partially supported by European Commission through Project Contract No. NMP4-CT-2003505311 (NABIS) and No. IST-500120 (NAPA), and by grants from the DGA and the French Embassy in China. One of the authors (I. Tapsoba) thanks l’Agence Universitaire de la Francophonie (AUF) for his postdoctoral fellowship. References [1] B.G. Chung, L.A. Flanagan, S.W. Rhee, P.H. Schwartz, A.P. Lee, E.S. Monuki, N.L. Jeon, Lab. Chip 5 (2005) 401. [2] N. Futai, W. Gu, J.W. Song, S. Takayama, Lab. Chip 6 (2006) 149. [3] Y. Jiang, B.N. Jahagirdar, R.L. Reinhardt, R.E. Schwartz, C.D. Keene, X.R. Ortiz-Gonzalez, M. Reyes, T. Lenvik, T. Lund, M. Blackstad, J. Du, S. Aldrich, A. Lisberg, W.C. Low, D.A. Largaespada, C.M. Verfaillie, Nature 418 (2002) 41. [4] M. Le Berre, C. Crozatier, G. Velve´ Casquillas, Y. Chen, Microelectron. Eng. 83 (2006) 1284. [5] P. Bourin, L. Sensebe´, P. Charbord, He´matologie 10 (2004) 434.
Microfluidic modulus for convenient cell culture and screening experiments C. Crozatier b
a,*
, I. Tapsoba a, L.P. Xu b, D. Han c, L. Sensebe´ d, Y. Chen
a
a Ecole Normale Supe´rieure, CNRS-UMR 8640, Paris 75005, France Centre for Microfluidic and Nanotechnology, Peking University, 100871 Beijing, China c National Centre for Nanoscience and Technology, 100080 Beijing, China d Etablissement Franc¸ais du Sang Centre-Atlantique, Tours 37020, France
Available online 15 February 2007
Abstract We report on a microfluidic set-up for convenient biological experiments involving cell culture. The strategy is to use a patterned microfluidic cover plate, which can be reversibly bonded to a cell culture substrate, to achieve both long-term cell culture in parallel chambers and control of culture parameters in each chamber. The design is furthermore optimized to enable intrinsic flow rate control. Preliminary results indicate that this novel cells-on-chips concept may lead to the development of microfluidic screening experiments and bio-analysis based on more conventional and high performance observations. 2007 Elsevier B.V. All rights reserved. Keywords: Cell culture; Microfluidics; Screening; Reversible assembly
1. Introduction Microfluidics is a new scientific discipline which is particularly relevant for biological experiments. This is because of the fact that most of the traditional biological experiments are time consuming and laborious. It is highly desirable to develop integrated systems that allow high throughput screening and/or automatic processing of a sequential experimental steps. The concept of ‘‘lab on a chip’’ microfluidic devices fully fits these requirements. In addition, it provides great advantages in the economy of chemical reagents and biological samples. In fact, on-chip cell culture and differentiation studies [1] as well as high throughput cell culture assays [2] have been developed, but they have yet to be compiled and adapted to the precise needs of cell biologists. The main difficulties for such exploration are the fluid control over a long period of time and the fragility of manipulated objects, where slight changes in the parameters can lead to death of all the sam*
Corresponding author. Tel.: +33 1 44322421; fax: +33 1 44322402. E-mail address: [email protected] (C. Crozatier).
0167-9317/$ - see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.mee.2007.01.265
ples. Most of the microfluidic chips developed so far were based on irreversible bonding and hydrostatic pressure or mechanic pumping for fluid control [3]. With these types of cell chips and optimised protocols, it is possible to put a cell population in one stable culture or differentiation condition. However, two issues arise for advanced studies: how to develop screening experiments with parallel and precise fluid control with numerous entries and how to adapt the common used bio-analytical systems to the ‘‘lab on a chip’’ applications. We hereby address these issues by proposing a novel microfluidic modulus that enables inherent flow control through its design and that conveniently gives access to the biological samples after experiments. 2. Experimental 2.1. PDMS device fabrication Soft lithography has been used to fabricate microfluidic devices. With a high resolution (3600 pixel per square inch) laser printer, a transparent plastic film was patterned as
C. Crozatier et al. / Microelectronic Engineering 84 (2007) 1694–1697
mask for contact printing optical lithography. The designed fluidic channels and the patterns for the stamps were then replicated into a 30 lm layer of AZ-9260 photo-resist deposited on a silicon substrate by UV light exposure. Before casting, the resist patterns were exposed to trimethylchlorosilane (TMCS) vapour for 1 min to aid subsequent release. Then, the liquid pre-polymer PDMS was poured on and cured at 80 C for 1 h. After peeling off the cross-linked PDMS structures, if necessary, holes were punched through the elastomer to allow later fluidic access to the reservoirs. The microfluidic device is then washed with ethanol and dried under filtered air flow. No specific treatments were done unless stated otherwise. 2.2. Reversible sealing of PDMS devices with microaspiration A new method has been used to make functional microfluidic channels that can be peeled off from the cell culture substrate [4]. Briefly, in order to realize reversible sealing between the microfluidic channels and the substrate, a network of crossing channels of air has been designed to surround the channels (see Fig. 1). This results in a cavity where vacuum of around 150 mbar can be applied with the help of a mini pump. Such aspiration gives enough strength to hold the microfluidic structure layer and the substrate together with a good sealing, hence allowing various manipulations on the device such as changes of outlets or reservoirs. In addition, the fact that PDMS is permeable to air enables the sucking out of air bubbles trapped in the microfluidic channels. This leads to an easy filling and the certainty that each device is ready for cell culture. Stopping the aspiration will enable the disassembly of the microfluidic device from the substrate. 2.3. Cell culture Rat mesenchymal stem cells were used for cell culture. All details about culture medium and traditional culture
Fig. 1. A schematic figure showing the microfluidic (A) and vacuum (B) channels. The vacuum channels (B) are interconnected, and when vacuum is applied (lower figure), the overall PDMS structure is deformed clamped onto the substrate.
1695
protocols are described elsewhere [4]. Here, the substrate for cell culture is a glass slide coated with Fibronectin (Sigma), a cell adhesion promoter protein, at 4 C over night. Once the glass slide is rinsed and dried, the microfluidic modulus is applied directly onto it. This step must be done in a dust-free environment to prevent leakage. The device is then filled with culture medium by applying droplets of medium at each inlet and outlet and by producing vacuum in the network of crossing channels. The air in the microchannels are then evacuated through the channel walls. Once the cells are loaded into the chambers, a static period of a few hours is required for them to start adhering to the substrate. Afterward, a flow of fresh culture medium is applied at around 100 nL/min to enable cell proliferation.
3. Results and discussion 3.1. Long-term stable flow A stable flow of culture medium in a chamber has proven to be crucial for reliable cell culture protocol. Cells are delicate and complex objects that evolve, quite often unpredictably, with the slightest change in their environment. The shear stress suffered by the cells is quite high because of the dimensions of the chambers (30 lm high). We have conveniently included in the design of the chip an inherent flow control part consisting of a series of channels with outlets in between. Applying an outlet pressure at a different point will result in variation of the overall hydrodynamic resistivity. Two advantages can be taken from this set up. First, we have added overall resistivity to the microfluidic device, hence permitting both better reduction of the variability in the flow rate caused by inherent differences between chambers, and the use of a more stable and convenient flow control device for long-term experiments. In addition, we are now able to regulate the flow rate as we see fit only by choosing the device outlet. This circumvents drastic changes in the predicted flow rate caused by fabrication errors since these unpredictable factors can now be taken into account and the flow rate adjusted in consequence. A vacuum pump has been chosen as a flow control device since it is convenient and stable through time. A vacuum of 300 mbar has been applied at the outlet in order to have a stable underpressure throughout the whole experiment. Too little vacuum is very difficult to stabilize because any slight variation, in the reservoirs’ volume for example, can dramatically change the applied difference of pressure. Here, the overall resistivity of the device was tuned with numerous outlets put into series at the end of the initial one. Each outlet is separated by a microchannel of 100 lm wide and 1 cm long and each is linked to a clamped tube when unused. Fig. 2 shows the variation of the flow rate resulting from the change of outlet: a stable flow with a flow rate as slow as 3 nL/s (corresponding to the 100 nL/
1696
C. Crozatier et al. / Microelectronic Engineering 84 (2007) 1694–1697
Fig. 3. Microphotographs of cell culture in microfluidic chambers: left: cell attachment after seeding; right: cell proliferation after ten days of medium flow.
Fig. 2. Average flow rate variation due to the variation of the overall resistivity of the microfluidic device. Between each output, a channel of 30 lm · 100 lm · 1 cm was added to increase the overall resistivity (see above).
min rate used in our usual on chip cell culture experiments) has been achieved. 3.2. Long-term cell culture On chip cell culture experiments have been described in numerous configurations, but the use of micro-aspired devices is unprecedented. As described in the experimental section, the micro-aspiration technique enables the reversible bonding of the device to the substrate, hence facilitating the retrieval of biological samples once the screening has been done. Moreover, the micro-aspiration technique has proven to be convenient of use for bubble suppression as well. Indeed, screening experiments are cumbersome especially for differentiation parameter screening because of the quantity of conditions to be tested. This has also an impact in microfluidic devices since the number of inlets and the risk of bubble formation increase: a simple bubble can alter the functioning of the whole device. Because a depression is applied all around the microfluidic channels and because the material of the device is made of, PDMS, it is permeable to gas, incomplete filling and bubble formation are no longer of concern. With these advantages in the micro-aspiration technique, it was possible to realize long-term cell culture in parallel chambers. Fig. 3 shows a usual result of an on-chip cell culture experiment where cell proliferation can be observed. Fig. 4 (left) shows one of the microfluidic devices used for parallel cell culture experiments. Each culture chamber was linked to a reservoir that could be filled at will with different substances. Fig. 4 (right) shows a microphotograph of cells in a chamber.
Fig. 4. Left: photography of the long-term cell culture parameters screening device. Right: a microphotography of cells cultivated in a microchamber.
3.3. General applications This device has initially been developed for screening the parameters in the differentiation pathway from MSC stem cells to neural cells [5]. Fig. 5 shows an example of such a differentiation pathway, already tested in one of the
Fig. 5. Left: testing cell differentiation protocol: after each incubation period, the surrounding medium is replaced with other transcription factors. The timing of these changes in the medium is crucial. Here, both SHH and FGF8 factors will vary in concentration. These proteins are crucial for inducing cells into specific differentiation pathways. Right: Immunocytochemistry of rMSC cells engaged in neural differentiation pathway (red: expression of Nestin, a neural protein not expressed in usual rMSC cells; green: fluorescent dyeing for cells; bottom: superposition of both fluorescent image to show that some cells express typical neural proteins, see yellow-colored cells). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
C. Crozatier et al. / Microelectronic Engineering 84 (2007) 1694–1697
authors’ laboratories. With the possibility to add pumps, valves and mixers into a micro-aspired chip, it is now possible to realize a more sophisticated microfluidic modulus that can become an essential tool for screening experiments. Moreover, as there is no restriction for the substrate used with the microfluidic modulus, as long as it is smooth and planar across the aspiration network, we can couple this experiment with analytical devices such as protein or DNA chips for in situ analysis. 4. Conclusion We have successfully applied a novel strategy for onchip microfluidic techniques integration in biological experiments. Cell loading and attachment could be achieved using a substrate that can be subsequently treated and analyzed with traditional methods. Except for the microfluidic device itself, manipulations require only basic materials that can be found in common biological laboratories (tubes, syringes, microscope and vacuum pump). These screening experiments can therefore be performed in a standard cell biology environment. We believe that this
1697
strategy is relevant to achieve a better efficiency in various biological screening experiments. Acknowledgements This work was partially supported by European Commission through Project Contract No. NMP4-CT-2003505311 (NABIS) and No. IST-500120 (NAPA), and by grants from the DGA and the French Embassy in China. One of the authors (I. Tapsoba) thanks l’Agence Universitaire de la Francophonie (AUF) for his postdoctoral fellowship. References [1] B.G. Chung, L.A. Flanagan, S.W. Rhee, P.H. Schwartz, A.P. Lee, E.S. Monuki, N.L. Jeon, Lab. Chip 5 (2005) 401. [2] N. Futai, W. Gu, J.W. Song, S. Takayama, Lab. Chip 6 (2006) 149. [3] Y. Jiang, B.N. Jahagirdar, R.L. Reinhardt, R.E. Schwartz, C.D. Keene, X.R. Ortiz-Gonzalez, M. Reyes, T. Lenvik, T. Lund, M. Blackstad, J. Du, S. Aldrich, A. Lisberg, W.C. Low, D.A. Largaespada, C.M. Verfaillie, Nature 418 (2002) 41. [4] M. Le Berre, C. Crozatier, G. Velve´ Casquillas, Y. Chen, Microelectron. Eng. 83 (2006) 1284. [5] P. Bourin, L. Sensebe´, P. Charbord, He´matologie 10 (2004) 434.