Continuous perfusion of mammalian cells embedded in agarose gel threads

Experimental Cell Research 154 (1984) 521-529

Continuous

Perfusion of Mammalian Cells Embedded in Agarose Gel Threads

DAVID L. FOXALL,‘,* JACK S. COHEN’, ** and JAMES B. MITCHELL2 ‘Laboratory of Physical and Theoretical Biology, National Institute of Child Health and Human Development and ‘Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20205, USA

A method for perfusing cells by embedding them in tine agarose gel threads is described and characterized. The rate of diffusion of a metabolite into the gel threads is determined by 3’P-NMR spectroscopy. This perfusion method is shown to enable Chinese hamster lung tibroblasts (CHLF) to remain in a metabolically active state with high levels of intracellular ATP for many hours.

We have recently given a preliminary description of a method for perfusing microbes, which involves embedding them within agarose gel cast in the form of fine threads [l]. The packed gel threads are then perfused by passing a culture medium through them. In this paper we discuss the progress we have made in characterizing this method of perfusion and describe its application to the study of mammalian cells. The cell lines employed in our study were cultured Chinese hamster lung fibroblasts (CHLF). As a test of the efficiency of diffusion of metabolites into the gel matrix we have directly measured the diffusion of ATP into plain threads by 31P-NMR spectroscopy. The results of these experiments are interpreted with a theory based upon the solution of the diffusion equation for a long cylinder [2]. We have also investigated the possibility that the morphology of the CHLF cells could be altered during the process of fabricating the gel threads by comparing cells in the gels with cells in free suspension. Further and conclusive tests of the gel-thread perfusion method involved monitoring the metabolic state of embedded hamster fibroblasts non-invasively by “P-NMR spectroscopy [3-51. Two experiments were carried out, the first simply to find the length of time the cells remained metabolically stable in the gel threads when perfused with a fixed volume of medium. In the second experiment the cells were perfused with medium containing 2-deoxyglucose in place of glucose to investigate the uptake of metabolites from the perfusion medium and their utilization within the cell. * Current address: Clinical Pharmacology Branch, NCI, NIH, Bethesda, MD 20205, USA. ** Current address: Varian Associates Inc., Palo Alto, CA 94304, USA. Copyright @ 1984 by Academic Press, Inc. All rights of reproduction in any form reserved W14-4827/84 so3.00

522 Foxall, Cohen and Mitchell b

/Teflon

Tubing Reservoir

“0”

ii T&Ion Tube

(0.5 m-n id)

,Screv-On

Rings

cap

Air Pressure frcm Pump

Cell Growth seal Cap Water Bath 37’ Liquid Gel Cell Mixture

Gel-Cdl Threads

1

Geccell Thread

t

Fig. 1. (a) Block diagram of the apparatus required to embed cells within threads of agarose gel. A

mixture of liquid agarose and cells is extruded through a fine Teflon tube chilled in ice. The solid thread formed within the tine tube is then collected directly into a 10 mm NMR tube and is ready for perfusion. (6) Schematic of the modified NMR tube used for perfusing the gel thread matrix during NMR experiments.

EXPERIMENTAL Cell Culture CHLF cells were grown in F12 medium supplemented with 10% fetal calf serum (FCS), penicillin, streptomycin, and Hepes buffer (20 mM). Since these studies required significant quantities of cells, these were grown in exponential growth in 850 cm2 roller bottles maintained at 37°C. Cells were harvested from the roller bottles by trypsin treatment, rinsed, and resuspended in growth medium to a final concentration of ca 10scells/ml for gel thread preparation. For all studies the pH of the medium was maintained at 7.3; only exponentially growing cells were used, and the plating efftciency as measured by macroscopic colony-forming ability ranged between 80 and 95%. Cell survival was determined after a certain perfusion period in the gel threads by gently pipetting the cells free of the gel, counting and plating a known number of cells for colony-forming ability. Following a 7-IO-day incubation period, colonies were fixed, stained and counted.

Preparation of Gel Threads Fabrication of the gel-thread matrix containing V79 CHLF cells was carried out as follows. A solution of low gelling temperature (LGT) agarose (1.8 % w/v) was prepared in a balanced salt medium (CaC12,0.9 mM; MgC12, 0.5 mM; KCl, 2.7 mM; NaCI, 137 mM; glucose, 10 mM; and Hepes, 10 mM, pH 7.4) and was stored in the liquid state at 37°C until required. Cells in their growth medium were mixed with the agarose solution in a 2 : 1 ratio so that a typical sample contained 2-3 x 10’ cells in 1.8 ml of 0.6 % agarose. The gel matrix was then extruded under mild pressure through a coil of teflon tubmg (0.5 mm i.d.) chilled in an ice bath. The apparatus used to prepare the threads is illustrated in fig. 1a. The gel threads containing the cells were then collected directly into medium in a tube. Plain Erp Cell Res 154 (1984)

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agarose threads for studies of diffusion were prepared without cells from a 0.6% solution in LGT agarose in 0.1 M phosphate buffer (pH 7.4), using capillary tubing of different internal diameter as required.

Per&ion The apparatus used for perfusion is shown in fig. 2, the cell threads being contained in a compartment at the bottom of a 10 mm screw cap NMR tube (Wilmad, Inc.). The insert and connections were fabricated from Kel-F. Samples of gel matrix containing cells were perfused with F12 growth medium supplemented with either glucose or 2-deoxyglucose at a concentration of 20 mM. The flow rate of perfusate was 2.5 mhmin. The plain agarose threads used in the diffusion studies were perfused at a flow rate of 2.9 ml/min with 0.1. M phosphate buffer (pH 7.4) to which ATP (0.1 M) was added. The volume of the perfusate was generally kept to 50 ml, and the reservoir was efficiently mixed to ensure that all reagents present in the perfusate were uniformly distributed. In perfusion studies with cells all precautions were taken to ensure sterility, including autoclaving the apparatus, rinsing with ethanol followed by aqueous penicillin and streptomycin (0.2 g each in 200 ml). In addition the perfusate was changed every 2-3 h to avoid depletion of any component and the growth of bacteria.

Light Microscopy Photomicrographs of cells and gel threads were taken with a Nikon inverted phase contrast photomicroscope to determine the morphologicat state of the cells.

NMR Spectroscopy “P-NMR spectra were obtained at 109.3 MHz using a Nicolet NT270 spectrometer as described elsewhere [l]. During time course experiments spectra were accumulated sequentially and then stored on disk under computer control.

RESULTS AND DISCUSSION Diffusion

in the Gel Threads

The successful perfusion of cells entrapped within gel fibres depends upon the supply of nutrients diffusing into the gel. The diffusion of nutrients becomes more efficient as the diameter of the libres decreases. On the other hand, the ease with which the gel threads can be cast and manipulated increases with their diameter. The optimum choice for the thickness of the threads is therefore a compromise between their mechanical strength and their diffusion properties. The determination of the optimum dimension for the gel threads to be used in the perfusion system with cells was investigated by studying the diffusion of ATP into plain threads of different diameters. ATP was chosen simply because it was a convenient substance to observe by “P-NMR spectroscopy. Changes in the ATP content of the gel were followed by monitoring the intensity of the “P-NMR signals from ATP as a function of time following addition of ATP to the perfusion reservoir. The intensity of the NMR signal from the sample during the perfusion of plain gel threads with ATP is given by

(1) Exp Cell Res I54 (1984)

524 Foxall, Cohen and Mitchell

13

b

,I

140

Fig. 2. Stacked plots of the “P-NMR

spectra obtained during the time course of perfusion for plain gel threads. The intensity of the ATP signals depend upon its concentration within the gel, while the intensity of the resonance from phosphate remains constant as this species was at equilibrium in the gel-buffer system. Plot a was obtained using 2 mm diameter threads; spectra were accumulated at 3 min intervals. The initial rise in the ATP signal corresponds to the dead time taken to flush the system with buffer containing ATP, with a dead volume of 0.84 ml and a flow rate of 2.9 ml min-’ the dead time is approx. 17 sec. Plot b was obtained using 0.5 mm diameter threads and spectra were obtained every 20 sec. The ATP reaches equilibrium in 80 sec.

were, S, is the equilibrium value of the NMR signal, COis the concentration of ATP in the pet&sate, and Cg(t) is the average concentration of ATP in the gel; VT, V, and V, are the volumes of the sample, the gel and the space occupied by the perfusate, respectively. The equation which describes diffusion into a long cylinder has been solved (2), and the average concentration of molecules in the gel threads can be shown to be

C,(t) = CO I- 2 d//3, exp ( Exp Cd

n=l

Res 154 (1984)

(-D(BJa)*f) >

Cell perfusion

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Fig. 3. (a) Phase contrast micrograph of agarose threads containing CHLF cells; (b) CHLF cells in free suspension; (c) photomicrograph of CHLF cells embedded in a gel thread. (a) x100; (b, c) x250.

where a is the radius of the threads, D the diffusion coefficient in the gel phase and /I,, the nth root of the Bessel function Jo(x). The results of the ATP perfusion experiments are shown in fig. 2. The diffusion time course for 2 mm diameter threads was fitted to the overall expression for the NMR signal intensity, and a value of 1.37+0.04x 10m6cm2 set-’ was obtained for the diffusion coefficient of ATP in agarose. This value is in the general range expected for diffusion coefficients in agarose gels, which are typically 30-50% of the corresponding value in water [6]. As a reasonable goal it was decided that metabolites should diffuse into the threads and come to equilibrium in less than 3 min. Calculations based on the diffusion coefficient of glucose and other molecules in agarose indicates that 0.5 mm threads will allow diffusion to establish equilibrium within the gel in this Exp Cell Res 154 (1984)

526 Foxall,

Cohen and Mitchell

Fig. 4. “P-NMR spectra of a dense suspension (ca 108cells/ml) of CHLF cells. These spectra show the loss of signals from ATP and ADP (a-c) and of the sugar phosphates (4. The growth and shift of the phosphate resonance (e) indicates that the cells have depleted their nutrient supply and have become acidic.

period of time. The results of the ATP perfusion experiment with 0.5 mm diameter thread (fig. 26) indicates that this goal was realised in practice, since equilibrium was established in 80 sec. Morphology

of Cells in Gel Threads

A phase contrast microscope picture of the gel threads reveals that the threads are essentially a matrix of closely packed cells (fig. 3 a). Phase contrast microscope pictures of the cells near the surface of the gel threads or teased out by gentle shaking were essentially the same as those obtained with these cells in free solution (fig. 3 b, c). Thus, it appears that embedding in the gel threads had no obvious observable effect upon the morphology of the CHLF cells. In addition to these observations sections of threads fixed in a paraffin block and stained indicated that the cells were essentially uniformly distributed throughout the threads and appeared morphologically normal. Perfusion

with Cells in the Gel Threads

31P-NMR may be used as a non-invasive technique to monitor the major (>l mM) phosphorylated metabolites within cells. Using 31P-NMR it is generally found that if cells are observed in dense suspension the ATP signals decrease to zero over a period of l-2 h (fig. 4). Consequently any metabolic event that is Exp Cell Res 154 (1984)

Cell perjhion

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Fig. 5. A stacked plot of the 3’P spectra obtained during the time course of perfusion for CHLF cells embedded in agarose threads. The spectra were obtained every 20 min over a period of 4 h. The levels of ATP remain constant during the whole time course indicating that the cells were able to obtain sufficient nutrients to maintain their metabolism.

being observed is also being seen against this background. In many cases, by the time the cells are harvested, centrifuged, and packed in dense suspension no signal other than Pi is observable. We tested the capability of the gel-thread perfusion system to maintain cells in a metabolically stable state for as long as possible. The perfused CHLF cells retained their ATP levels for up to 24 h (fig. 5), provided all precautions were taken as described above, including changing

60mins

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Fig. 6. A time course of spectra from CHLF cells in agarose gel threads following the addition of 2deoxyglucose (20 mM) to the perfusion medium. The growth of (a) intracellular 2-deoxyglucose 6phosphate; (b) AMP are seen. Exp Cd

Res 154 (1984)

528 Foxall, Cohen and Mitchell the perfusate every few hours. This result was very reproducible, and indicates that the gel-thread perfusion method has the capability to enable extensive noninvasive metabolic studies by NMR methods over periods of many hours. In addition, if cells that have been perfused for 24 h are gently pipetted free of the gel threads and plated for colony-forming ability, the plating efficiency ranged from 50 to 75 %, thus indicating the flexibility for non-invasive metabolic studies and subsequent cell survival studies using the same initial cell population. The second test of the perfusion system is to demonstrate that the cells respond to an added reagent, indicating that it diffuses into the threads and the cells. For this purpose we utilized 2-deoxyglucose which we had previously used with yeast [I]. This analog of glucose is known to be transported across the cell membrane, but is not metabolized further once phosphorylated. Upon the addition of 2deoxyglucose to the perfusate the “P signals of 2-deoxyglucose 6-phosphate and AMP began to increase in ca 20 min and the level of ATP gradually decreased (fig. 6). This corresponds to previous experience with this metabolite [l, 71, consequently it appears that the gel-thread perfusion system provides an excellent basis for the investigation of cell metabolism [8]. We are currently investigating the response of CHLF cells to hyperthermia treatment and observing the 3’PNMR spectra of a variety of cancer cells. Neither of these projects were found to be feasible in simple cell suspensions, but require the ability to perfuse for several hours to obtain meaningful results. General Significance of the Perfusion Method While the perfusion technique described herein was developed primarily to facilitate non-invasive NMR studies of cell metabolism, it would appear to have a broader range of applicability. Other methods of trapping cells in gel have been described, and these methods have been used to isolate cell secretion products. Thus, in one method cells were trapped inside agarose beads made by stirring cells and agarose in an oil suspension [9]. The potential applications of our gel-thread method to both the isolation of cellular products and the division of cells within a perfused matrix other than purely agarose warrant further investigation. We thank William Hagins for help with the cell photomicrographs.

REFERENCES 1. Foxall, D H & Cohen, J S, J mag res 52 (1983) 346. 2. Barrar, R M, Diffusion in and through solids. Cambridge University Press, Cambridge (1951). 3. Shulman, R G, Brown, T R, Ugurbil, K, Ogawa, S, Cohen, S M &den Hollander, J A, Science 205 (1979) 160. 4. Gadian, D G, Nuclear magnetic resonance and its application to living systems. Clarendon Press, Oxford (1982). 5. Cohen, J S, Knop, R H, Navon, G & Foxall, D H, Life them rep 4 (1983) 281. 6. Friedman, L & Kraemer, E 0, J Am them sot 52 (1930) 1295. Exp Cell Res 154 (1984)

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7. Navon, G, Ogawa, S, Shulman, R G & Yamane, T, Proc natl acad xi US 74 (1977) 87. 8. Knop, R H, Chen, C W, Mitchell, J B, Russo, A, McPherson, S & Cohen, J S, Biochim biophys acta (1984). In press. 9. Nilsson, K, Scheirer, W, Merton, 0 W, Ostberg, L, Liehl, E, Katinger, H W D & Mosbach, K, Nature 302 (1983) 629. Received March 14, 1984

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Exp Cell Res 154 (1984)