Biomacromolecules 2003, 4, 1558-1563
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Enzymatic Methods for in Situ Cell Entrapment and Cell Release Tianhong Chen,†,‡ David A. Small,†,§ Martin K. McDermott,| William E. Bentley,†,§ and Gregory F. Payne*†,‡ Center for Biosystems Research, University of Maryland Biotechnology Institute, 5115 Plant Sciences Building, College Park, Maryland 20742-4450, Department of Chemical and Biochemical Engineering, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250, Department of Chemical Engineering, University of Maryland at College Park, College Park, Maryland 20742, and Division of Mechanics and Materials Science, Office of Science and Technology, Food and Drug Administration, 9200 Corporate Blvd., HFZ-150, Rockville, Maryland 20850 Received May 12, 2003; Revised Manuscript Received July 30, 2003
We report an enzyme-based method for the in situ entrapment of cells within a biopolymeric hydrogel matrix. Specifically, we used a calcium-independent microbial transglutaminase that is known to cross-link proteins and observed that it catalyzes the formation of gels from a pre-gel solution containing 10% gelatin and E. coli cells. Hydrogel formation occurs 2-3 h after adding transglutaminase, and no additional external intervention is required to initiate gel formation. The in situ entrapped cells grow rapidly and to high cell densities within the gelatin hydrogel. Additionally, the entrapped cells respond to isopropylthiogalactoside induction. The cross-linked gelatin network can be rapidly hydrolyzed (within 1 h) by the protease, proteinase K. Treatment of the network by this protease releases the entrapped E. coli cells. These cells appear unharmed by proteinase K; they can grow and be induced after protease treatment. The ability to in situ entrap, grow, and release cells under mild conditions provides unique opportunities for a range of applications and should be especially useful for microfluidic biosensor systems. Introduction There is a growing interest in in situ methods for hydrogel formation.1-3 For instance, the creation of cell-based microfluidic biosensors would be facilitated if methods were available that allowed a cell-containing pre-gel solution to be mixed and transported through a microfluidic network to some location where hydrogel formation could be initiated. The ideal method for such applications would meet the following criteria: all ingredients are benign to cells; the pre-gel solution state has a low-viscosity to facilitate transport; the gels form only when/where they are desired while requiring a minimum of external manipulations; and the gel’s properties are spatially homogeneous and adjustable (e.g., by varying the composition of the pre-gel solution). Currently, the in situ methods that appear best-able to meet these criteria employ the photoinitiation of a pre-gel solution. Such in situ photoinitiated hydrogel-forming methods are being investigated for both biosensor4-6 and tissue engineering7-12 applications. In the current study, we examine an alternative method for in situ hydrogel formation. Specifically, we use a calcium* To whom correspondence should be addressed. E-mail: payne@ umbi.umd.edu. Phone: (301) 405-8389. Fax: (301) 314-9075. † University of Maryland Biotechnology Institute. ‡ University of Maryland Baltimore County. § University of Maryland at College Park. | Food and Drug Administration.
independent, microbial transglutaminase enzyme that catalyzes trans-amidation reactions that introduce N -(γglutamyl)lysine cross-links in proteins.13 We use this enzyme to convert the protein gelatin into a three-dimensional, covalently cross-linked hydrogel network. Previous studies have shown that this enzyme can covalently cross-link gelatin to create a “chemical” gel.14-18 These cross-links are permanent, and the chemical gels cannot be “melted” by heating above gelatin’s helix-to-coil transition temperature.16 Thus, the transglutaminase-catalyzed chemical gel is different from the thermally responsive “physical” gel characteristic of gelatin.
The first goal of this work was to determine if transglutaminase could be used as an in situ method to entrap cells while retaining their viability and sensing capabilities. Our second goal was to determine if the entrapped cells could be released by protease treatment. To demonstrate these concepts, we used an E. coli strain that expresses green fluorescent protein (GFP) from an isopropylthiogalactoside (IPTG), inducible promoter.19,20 This model biological system allows growth and induction to be readily measured using simple, optical methods.
10.1021/bm034145k CCC: $25.00 © 2003 American Chemical Society Published on Web 09/30/2003
Methods for in Situ Cell Entrapment and Release Scheme 1
Materials and Methods Gelatin (type A from porcine skin, 175 bloom), proteinase K (from Tritirachium album), and IPTG were purchased from Sigma Chemicals. Ampicillin was obtained from FisherBiotech. The calcium-independent, microbial transglutaminase was obtained from Ajinomoto and was the same batch as used previously21 (Activa TI, reported by the manufacturer as 100 U/gm based on the hydroxylamine reaction,13,16 lot number 97.12.04). We report the lot number because of the batch-to-batch variability of this enzyme. All materials were used without further purification. The E. coli BL21/pTrcHisBGFPuv transformant used in this study was reported elsewhere.19,20 Briefly, E. coli BL21 was transformed with the ampicillin-resistant plasmid pTrcHisB that had been modified to express a hexahistidinetagged green fluorescent protein (GFP). Expression was regulated by an IPTG-inducible, trc promoter. The experimental methods for this study are illustrated in Scheme 1. A total of 15 mL of Luria Broth (LB) medium (5 g/L yeast extract, 10 g/L tryptone, and 10 g/L NaCl) with ampicillin (60 mg/L) was used to culture the cells. To generate gels, gelatin (10 w/v %) and transglutaminase (20 units/gm gelatin) were included in the medium. The initial medium pH was adjusted to 7 and all ingredients except transglutaminase were filter sterilized (0.2 µm) prior to inoculation. Cells were cultured in Petri dishes to facilitate oxygen mass transfer. The Petri dishes were mildly shaken (100 rpm) to provide mixing of the pre-gelled solutions while limiting spillage. All experiments were performed at 37 °C. The in situ gel formation of gelatin was monitored using Thermo Haake RS1 rheometer with a parallel plate sensor (PP60 Ti) and a gap distance of 1 mm. Gelatin (10 w/v % final concentration) was dissolved into the LB medium at a temperature of about 60 °C and the pH was adjusted to 7. This solution was then filter sterilized and ampicillin (60
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mg/L final concentration) was added. Transglutaminase (20 Units/gm gelatin) was added to this solution, and a sample of this solution was immediately loaded onto the rheometer. The measurement of the sol-to-gel conversion was conducted at 37 °C. The rheometer was programmed to perform oscillatory measurements. During the first 5 h of this experiment, when the sample was a solution or weak gel, oscillations were performed continuously with a controlled stress of 0.5 Pa and a frequency of 0.1 Hz. When the sample had formed a stronger gel (after 5 h), oscillatory tests were performed intermittently with a controlled strain of 5% and a frequency of 0.1 Hz. The different measurement conditions were used in order to account for the different properties of the initial solution and the final gel.21 To limit damage to the evolving gel network, the rheometer’s parallel plates were never separated during the course of the experiment. Evaporation was reduced by covering the parallel plate sensor with a low viscosity silicon oil (S159-500, Fisher Scientific). The lower left branch in Scheme 1 shows that cells were induced after they had been entrapped and cultivated for 24 h. The gels were washed three times with LB medium (10 mL each time) and then induced by spreading an IPTGcontaining solution over the gel’s surface (the final IPTG concentration was 2 mM based on the total gel volume). After induction, the cells were incubated for another 24 h period. The lower right branch in Scheme 1 shows that cells were released by transferring 10 g of gel to a flask, treating the gel with proteinase K (100 mg/L), and incubating for 1 h with shaking (225 rpm). After this treatment, the gels were observed to be broken. IPTG (2 mM) was then added to the resulting liquid and the cells were incubated for an additional 24 h. The optical density was measured using MRX plate reader made by Dynex Technologies, Inc. GFP fluorescence was measured using a PerkinElmer LS55 luminescence spectrometer with an excitation and emission wavelengths of 395 and 509 nm, respectively. All photographs were taken using a Canon EOS D60 digital camera. The photomicrographs were taken using Olympus BX 60 microscope. Results Transglutaminase-Catalyzed Gel Formation. Rheological studies were performed to ensure that transglutaminase can convert gelatin solutions into a cross-linked network in the presence of LB medium ingredients. For this study, gelatin (10 w/v %) and LB medium were mixed, and then transglutaminase was added. Changes in the rheological properties of these samples were monitored using oscillatory measurements. Figure 1 shows that the elastic modulus (G′) remained low and nearly constant for the first 2 h of the experiment and then increased markedly. At about the third hour, G′ was observed to increase above the viscous modulus (G′′). The gel-point is often defined as the time when the elastic and viscous moduli cross. The results in Figure 1 are similar to those for the transglutaminase-catalyzed crosslinking of gelatin solutions21 and demonstrate that gel formation occurs in the presence of LB medium ingredients.
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Figure 1. Rheological evidence that transglutaminase catalyzes the sol-to-gel conversion. Gelatin (final concentration 10 w/v %) and LB medium with ampicillin (60 mg/L) were mixed at pH 7, and then transglutaminase (20 Units/gm gelatin) was added. Immediately after adding enzyme, a sample was loaded onto the rheometer, and the rheological properties were monitored using oscillatory measurements at 37 °C. To limit structural damage during the experiment, the sample was not removed from rheometer during the 24-h period of the experiment.
Figure 2. Growth curves for in situ entrapped E. coli cells. Cells were inoculated into 15 mL of filter-sterilized LB medium with gelatin (10 w/v %) and ampicillin (60 mg/L). Transglutaminase (20 Units/gm gelatin) was added to initiate gel formation. “Control Gels” were prepared as above but were not inoculated. The “Control Cells” were cultured in LB medium without gelatin and transglutaminase (i.e., this control remained a solution). All samples were incubated at 37 °C and Petri dishes were used for cultivation to facilitate oxygen transfer.
In Situ Cell Entrapment. To exploit transglutaminase for in situ cell entrapment, we prepared LB medium with gelatin (10 w/v %), inoculated this medium with E. coli and immediately added transglutaminase to the solution. Because of the potential for oxygen limitations in thick hydrogels, we cultivated these cells in Petri dishes. The “Control Gel” in Figure 2 was prepared as described above but was not inoculated. The results from this control demonstrate that the slight color changes that accompany gel formation do not significantly alter the optical density measurement. The “Control Cells” in Figure 2 were grown in a Petri dish with LB medium (without gelatin or transglutaminase). Figure 2 shows that the cells grew rapidly when the liquid medium was dispensed in Petri dishes. Figure 2 also shows the growth curve for cells that had been in situ entrapped within a gelatin hydrogel. Consistent with the results in Figure 1, the solutions were visually observed to form gels during the first 2 to 3 h of incubation. As illustrated in Figure 2, the in situ entrapped cells grew rapidly (albeit more slowly than the “Control Cells”) and reached the same final optical densities as the control. We should note that it was not possible to dilute gelled samples, and the highest optical density measurements in Figure 2 may be outside the region where optical density and cell
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Figure 3. Induction of in situ entrapped E. coli cells to express GFP. Cells were in situ entrapped and grown as described in the legend of Figure 2. After 24 h of cultivation, the gels were washed with LB medium (3 times with 10 mL each) and then induced with IPTG (2 mM based on total gel volume). The “Uninduced” control was entrapped, cultivated, and washed the same way but was not treated with IPTG.
Figure 4. Photographs of in situ entrapped and induced cells. The “Induced” and “Uninduced” control were treated as described in the legend of Figure 3. The “Control Gel” was treated as described in the legend to Figure 2. Gels were observed using (a) light or (b) UV illumination.
concentration are linearly related. In sum, Figure 2 shows that cells entrapped using transglutaminase retain viability and are able to grow extensively within the gel network. In the next experiment, we examined whether the in situ entrapped cells could be induced by IPTG. This experiment was performed as illustrated by the lower left branch of Scheme 1. After the first 24-h incubation, the gels were washed three times with LB medium to remove any loosely bound cells. Visual inspection indicated that few cells were removed from the gels during washing. After washing, the entrapped cells were induced with IPTG. Figure 3 shows that 2 h after adding IPTG the fluorescence began to increase, and it increased almost linearly until the 10th hour. The “Uninduced” control in Figure 3 shows little fluorescence increase. Thus, the results in Figure 3 demonstrate that the entrapped cells retain the ability to sense and respond to chemical stimuli (i.e., they can be induced by IPTG). Figure 4 shows photographs of in situ entrapped cells. The “Induced” sample in Figure 4a shows cells that were entrapped and cultivated for 24 h, then washed, induced with
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Figure 6. Effect of Proteinase K treatment on E. coli cell growth. Cells were inoculated in LB medium (15 mL) in Petri dishes and grown in liquid culture. Proteinase K was added to the “Protease Treated” sample after 2 h of incubation.
Figure 5. Photomicrographs of gel cross-sections with in situ entrapped and induced cells. The cells were treated as described in the legend of Figure 3. Cross-sections were viewed using (a) light or (b) UV illumination.
IPTG, and incubated for an additional 24 h (i.e., this procedure is illustrated by lower left branch in Scheme 1). The image using visible light shows these gels were turbid due to cell growth. When this “Induced” sample was illuminated with UV light, Figure 4b shows significant fluorescence. The turbidity of the “Uninduced” control in Figure 4a indicates that cells had grown within the gel matrix, whereas the limited fluorescence observed in Figure 4b indicates that little GFP had been produced in this uninduced sample. Figure 4 shows that the “Control Gel” lacks turbidity and fluorescence consistent with the absence of cells. Figure 4 provides a visual illustration of cell growth and induction for the in situ entrapped cells. Additionally, Figure 4 demonstrates that these in situ-formed gelatin gels allowed extensive cell growth without loss of gel structure. Figure 5 shows photomicrographs of the cross section of induced gels. Figure 5a shows that the E. coli cells are distributed throughout the gel network. When viewed using UV illumination, Figure 5b shows that cells within this network had been induced to produce GFP. These photomicrographs also suggest that the cell population and induction may decrease as a function of distance from the surface of the gel. Such a distribution in growth and response would not be surprising because of diffusional limitations for oxygen (and possibly IPTG) through the gel.22-25 Enzymatic Release of Entrapped Cells. In initial studies, we observed that proteinase K can hydrolyze the transglutaminase-catalyzed “chemical gels” of gelatin. To examine if this protease adversely affects E. coli cells, we inoculated
Figure 7. Proteinase K treatment “dissolves” transglutaminasecatalyzed gelatin gels. Experiments were performed as illustrated by the lower right branch of Scheme 1. After the initial 24-hour cultivation, 10 g of gel was transferred to a flask and treated with proteinase K (100 mg/L) and incubated with shaking (225 rpm) for 1 h. The “Protease-Treated” gel was converted to a solution while the “Untreated Gel” remained intact.
cells into Petri dishes containing liquid LB medium, incubated them for 2 h, and then added proteinase K. Figure 6 shows that the growth of cells exposed to proteinase K was similar to the growth of control cells that had not been exposed to this protease. Thus, proteinase K treatment does not suppress the growth of this E. coli. In the final set of experiments, we examined the use of proteinase K to release entrapped cells. The lower right branch of Scheme 1 shows the protocol for this experiment. After entrapping and cultivating the cells for 24 h, 10 g of gel was transferred to a flask, treated with proteinase K, and then incubated for 1 h with shaking. Figure 7 shows that after 1 h the proteinase K-treated gel had been converted to a solution, while the untreated gel remained intact. After 1 h of proteinase K treatment, the released cells were induced using IPTG and incubated for an additional 24 h. Samples from this proteinase K-released and IPTG-induced culture were centrifuged, the supernatant was discarded, and the pellets were photographed under UV illumination. Figure 8 shows that cells that were both released and induced had substantial GFP fluorescence. Figure 8 also shows that the pellet for an equivalent proteinase-K released but uninduced sample was not fluorescent. Thus, cells that had been entrapped with transglutaminase, grown in the in situ gelatin gel, and then released using proteinase K could be subsequently induced to produce GFP.
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Figure 8. IPTG induction of cells that had been released by proteinase K treatment. Experiments were performed as illustrated by the lower right branch of Scheme 1. After the 1 h proteinase K treatment, IPTG (2 mM) was added to the “Induced” sample and no IPTG was added to the “Uninduced” control. Both the induced and uninduced cells were incubated for an additional 24 h and centrifuged, the supernatant was discarded and the pellet was viewed using UV illumination.
Discussion A calcium-independent, microbial transglutaminase was used for the in situ entrapment of E. coli cells within a crosslinked gelatin-based network. From a biological standpoint, we believe this method has advantages compared to alternative methods for in situ entrapment. Because transglutaminase catalyzes gel formation by directly cross-linking gelatin, it is not necessary to add low molecular weight cross-linking agents (e.g., calcium or glutaraldehyde), initiators, or monomers. As a result, transglutaminase-catalyzed gel formation is expected to be relatively benign to cells compared to alternative in situ methods for gel formation. Additionally, because gelatin is nontoxic, this network and its break-down products are also expected to be benign to cells. Thus, it is not surprising that in situ entrapped E. coli cells grow rapidly and respond to inducer. From a procedural standpoint, the transglutaminasecatalyzed method differs from alternative in situ methods for cell entrapment based on photopolymerization, and each method has advantages. Transglutaminase-catalyzed gel formation requires no external intervention (after mixing the pre-gel solution) and occurs over the course of 2-3 h although the gel time can be modified somewhat by altering the conditions.21 This timing is convenient in the sense that cells can be thoroughly mixed and readily transported (pumped) to a desired location after which the gel network forms homogeneously. This procedure can be contrasted with photoinitiated hydrogels that require external stimuli to initiate gel formation and may yield networks with spatially varying properties. The procedural benefits of photopolymerization are that gel formation is fast, precise spatial control of gel formation can be achieved for thin gels, and illumination is easy to supply in some settings (e.g., in microfabrication facilities). The mechanical properties of the hydrogel’s network are very important for cell immobilization, although the most suitable properties may depend on the application. Elastic matrixes have been reported to be better-able to accommodate abrasions26 and possibly also to withstand the deformations associated with extensive cell growth. With
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respect to gelatin, previous studies indicate that cross-linking is necessary to obtain sufficient mechanical strength.6,27-29 To our knowledge, this is the first report of the use of transglutaminase to cross-link gelatin for cell entrapment. Importantly, we observed that this cross-linked gelatin network permitted extensive cell growth without noticeable damage to the gel. Further adjustments to the gelatin network’s mechanical properties can be made by adding other components (e.g., polysaccharides) to the matrix.17,21 The use of a protease to release cells from the gelatin matrix is directly analogous to the use of trypsin for releasing animal cells. This method is also similar to the use of “smart” surfaces that have adjustable abilities to bind cells.30-32 The cross-linked gelatin gels were broken by proteinase K within 1 h, whereas proteinase K treatment did not disrupt E. coli’s ability to grow or be induced. Thus, proteinase K provides a simple and rapid method to release cells from the gelatin matrix. In summary, we have shown that (i) transglutaminase can be used for the in situ entrapment of E. coli, (ii) the entrapped cells can be grown to high densities within the hydrogel matrix, (iii) the entrapped cells retain their sensing abilities, and (iv) protease treatment can release the cells from the matrix without suppressing their abilities to grow or be induced. We believe this enzymatic entrapment and release should provide unique opportunities for a variety of applications and especially for microfluidic biosensors.33 Acknowledgment. Financial support for this research was provided by the United States Department of Agriculture (2001-35504-10667) and the National Science Foundation (Grant BES-0114790). The authors would like to thank Eleanor Brown and Maryann Taylor from the United States Department of Agriculture for their helpful discussions. References and Notes (1) Elisseeff, J.; Anseth, K.; Sims, D.; McIntosh, W.; Randolph, M.; Langer, R. Proc. Natl. Acad. Sci. 1999, 96, 3104-3107. (2) Gutowska, A.; Jeong, B.; Jasionowski, M. Anat. Rec. 2001, 263, 342349. (3) Gerentes, P.; Vachoud, L.; Doury, J.; Domard, A. Biomaterials 2002, 23, 1295-1302. (4) Heo, J.; Thomas, K. J.; Seong, G. H.; Crooks, R. M. Anal. Chem. 2003, 75, 22-26. (5) Koh, W.-G.; Revzin, A.; Pishko, M. V. Langmuir 2002, 18, 24592462. (6) O’Connor, S. M.; Andreadis, J. D.; Shaffer, K. M.; Ma, W.; Pancrazio, J. J.; Stenger, D. A. Biosens. Bioelectron. 2000, 14, 871-881. (7) Bryant, S. J.; Anseth, K. S. Biomaterials 2001, 22, 619-626. (8) Cruise, G. M.; Hegre, O. D.; Scharp, D. S.; Hubbell, J. A. Biotechnol. Bioeng. 1998, 57, 655-665. (9) Halstenberg, S.; Panitch, A.; Rizzi, S.; Hall, H.; Hubbell, J. A. Biomacromolecules 2002, 3, 710-723. (10) Bryant, S. J.; Nuttelman, C. R.; Anseth, K. S. J. Biomater. Sci. Polym. Ed. 2000, 11, 439-457. (11) Elisseeff, J.; McIntosh, W.; Anseth, K. S.; Riley, S.; Ragan, P.; Langer, R. J. Biomed. Mater. Res. 2000, 51, 164-171. (12) Pathak, C. P.; Sawhney, A. S.; Hubbell, J. A. J. Am. Chem. Soc. 1992, 114, 8311-8312. (13) Motoki, M.; Seguro, K. Trends Food Sci. Technol. 1998, 9, 204210. (14) Fuchsbauer, H. L.; Gerber, U.; Engelmann, J.; Seeger, T.; Sinks, C.; Hecht, T. Biomaterials 1996, 17, 1481-1488. (15) Lim, L. T.; Mine, Y.; Tung, M. A. J. Food Sci. 1999, 64, 616-622. (16) Babin, H.; Dickinson, E. Food Hydrocolloids 2001, 15, 271-276.
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Biomacromolecules, Vol. 4, No. 6, 2003 1563 (26) Vogelsang, C.; Wijffels, R. H.; Ostgaard, K. Biotechnol. Bioeng. 2000, 70, 247-253. (27) Kuijpers, A. J.; Engbers, G. H. M.; Krijgsveld, J.; Zaat, S. A. J.; Dankert, J.; Feijen, J. J. Biomater. Sci. Polym. Ed. 2000, 11, 225243. (28) Svitel, J.; Curilla, O.; Tkac, J. Biotechnol. Appl. Biochem. 1998, 27, 153-158. (29) Ulubayram, K.; Aksu, E.; Gurhan, S. I. D.; Serbeti, K.; Hasirci, N. J. Biomater. Sci. Polym. Ed. 2002, 13, 1203-1219. (30) Ista, L. K.; Mendez, S.; Perez-Luna, V. H.; Lopez, G. P. Langmuir 2001, 17, 2552-2555. (31) Cunliffe, D.; Alarco´n, C. H.; Peters, V.; Smith, J. R.; Alexander, C. Langmuir 2003, 19, 2880-2887. (32) Ista, L. K.; Perez-Luna, V. H.; Lopez, G. P. Appl. EnViron. Microbiol. 1999, 65, 1603-1609. (33) Sundberg, S. A. Curr. Opin. Biotechnol. 2000, 11, 47-53.
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