fibroblast and hepatocyte behavior on synthetic polymer surfaces

Fibroblast and hepatocyte behavior on synthetic polymer surfaces

W. Mark Saltzman; Patricia Parsons-Wingerter, Kam W. Leong,’ and Shin Lint De par h e n ts of Chemical Engineering, Biomed ica 1 Engineering, and Bio phys ics, The John Hopkins University, Baltimore, Maryland 21218

Biodegradable poly(phosphoesters) with varying side group chemistry and copolymers of styrene and methyl vinyl ketone (MVK) with varying degrees of hydrophobicity were used to study the growth and behavior of surface-attached fibroblasts and hepatocytes. Mouse 3T3 fibroblasts and chicken embryo fibroblasts attached and proliferated on all of the polymers tested. Fewer cells attached to copolymers of styrene and MVK than to glass or tissue culture polystyrene controls; cell attachment to several poly(phosphoester) surfaces was indistinguishable from controls. The mean speed of fibroblast migration was faster on surfaces where fewer cells attached (59 to 84 pm/h on low attachment surfaces compared with 40 to 46 pm/h on high attachment surf aces). When surf ace-attached cells

were stained with fluorescently labeled phalloidin, only a fraction of the cells on low attachment surfaces were shown to have prominent arrays of actin filament bundles. Chicken hepatocytes also attached to the polymer surfaces. When a suspension containing a large number of cells was placed over the polymer surfaces, approximately 50% of the hepatocytes attached during the first 9 h. Surprisingly, hepatocyte attachment and viability in culture were relatively insen- sitive to the chemistry of the synthetic polymer substrates. Cell number increased by about a factor of 2 over the first 48 h of culture, then decreased back to -50% of initial cell number over the next several days. Cell morphology did depend on the chemical structure of the substrates.

IN TRODUC T ION

Most mammalian cells must be attached to a solid substrate or scaffold in order to proliferate’ and function.’ With the exception of hemopoietic cells, tumor cells, and gametes, this generalization applies both in vivo and in vitro. In the animal body most cells are supported by a tissue specific extracellular matrix (ECM)? When cells are cultured outside of the body, this chemical and mechanical support must be provided within the cell culture environment. Since many surfaces, including metals and polymers, could provide adequate mechanical support for attached cells, the differences in cell behavior following attachment to different surfaces must be related to physico- chemical properties of the surface.

*To whom correspondence should be addressed at Department of Chemical Engi- neering, The Johns Hopkins University, 3401 N. Charles Street, 42 New Engineering Building, Baltimore, MD 21218.

Journal of Biomedical Materials Research, Vol. 25, 741-759 (1991) 0 1991 John Wiley & Sons, Inc. CCC 0021-9304/91/060741-19$4.00

742 SALTZMAN ET AL.

Many important observations on the relationship between cell attachment and subsequent behavior have involved synthetic polymer surface^."^'^ So far, it has been difficult to predict or measure cell response to specific chemical groups present at the polymer surface. In fact, it is not clear whether properties of the surface affect cell attachment and behavior directly or indirectly through an absorbed layer of a secondary attachment molecule, such as a protein. Many attachment molecules, like fibronectin and laminin, have been identified in recent years and the effects of these factors on cultured cells have been examined.6 The molecular biology of some of these molecules is known in detail: but examination of their role in cell attachment and behavior is complicated. Even in the absence of exogeneous factors, cells will mod- ify their local environment by secreting additional attachment factors and inhibitors (e.g. proteases).

Almost all of the cell culture work performed today involves a limited number of polymer substrates. The most common cell culture surface is tissue culture polystyrene (TCPS): hydrophobic sheets of polystyrene are surface- modified to increase hydrophilicity by introduction of carboxyl and hydroxyl groups at the surface.8 In this report, we compare the behavior of cells on synthetic surfaces, where the structure of the polymer-and hence the properties of the surface- was systematically changed. We have examined the behavior of two types of anchorage-dependent cells: fibroblasts and hepatocytes.

MATERIALS AND METHODS

Polymer synthesis and surface preparation

Two classes of substrates, constructed from biodegradable polymers based on a poly(phosphoester) backbone and biostable polymers based on a vinyl backbone, were examined. Table I shows the chemical structures of all the polymers tested; abbreviations for each polymer are defined in this table. Poly(phosphoesters) with a bisphenol-A backbone and various side chains were synthesized by interfacial polycondensation conducted in CH2C12/ HzO with the aid of cetyl trimethylammonimum chloride as phase transfer catalyst.' To enhance the stability and vary the mechanical strength of the polymers, BPA/PP was also crosslinked with pentaerythritol. With low crosslinking, these polymers were still soluble in chloroform and thus could be spin-coated for cell culture studies. Our notation for these polymers, BPA/PP/6%, refers to the weight percent of pentaerythritol with respect to BPA; the exact crosslinking density has not been determined. In addition, copolymers of styrene and methyl vinyl ketone (MVK) of various composition were obtained by radical polymerization conducted in toluene at 60°C using azobisisobutyronitrile as the initiator. The styrene-MVK copolymers were washed extensively in distilled water before use. The chemical structures of all of the polymers were confirmed by FT-IR and FT-NMR. Water in air contact angles were determined by goniometry (Rame-Hart, Inc.). The weight average molecular weights of the poly(phosphoesters) were around

CELL BEHAVIOR ON POLYMERS 743

TABLE I Chemical Structures of the Polymers Tested

Polymer Chemical Structure

Poly(ethy1 phosphonic acid-bisphenol A) BPA/EP

Poly(ethy1 phosphate-bisphenol A) BPA/EOP

Poly(pheny1 phosphonic acid-bisphenol A) BPA/PP

Poly(pheny1 phosphate-bisphenol A) BPA/POP

Poly(styrene-co-methyl vinyl ketone) with X / ( X + Y ) mole fraction styrene

40,000 as determined by GPC. Typical values for the styrene-MVK copolymers were 6Or0O0 and 30,000 for the M , and M,, respectively.

Thin films of polymer on glass coverslips

Glass coverslips (45 x 50 mm, #2, Fisher Scientific) were cleaned by immersion in 1M HC1 overnight, immersion in IM NaOH for several minutes, and exhaustive rinsing with distilled water. Poly(phosph0esters) and vinyl polymers were prepared for cell culture studies by dissolving the selected polymer in methylene chloride (-10% w/v) and spin-coating (spin-coater I'M 101D-R485, Headway Research, Garland, TX) the polymer solution onto a clean glass coverslip. Polymer-coated coverslips were immediately placed in clean storage cases and stored desiccated at room temperature until use.

Design of polycarbonate cell culture chambers

In preliminary experiments, glass coverslips were placed in conventional TCPS culture wells and covered with cell suspension. Under these conditions

SALTZMAN ET AL. 744

cells attached on both the top and bottom surface of the coverslip; the slips were clearly floating on a film of f h id trapped between the coverslip and the plastic. To eliminate difficulties in interpreting subsequent experiments, cell culture chambers were machined from autoclavable polycarbonate. These chambers permitted a large number of parallel cell cultures, which were seeded with the same cell suspension on different polymer substrates.

Six holes of 35-mm diameter were cut with a minimum of 13-mm spacing into a solid rectangular sheet of 1.2-cm-thick polycarbonate (12 cm X 19 cm). Two adjacent corners of the polycarbonate rectangle were notched to permit rapid orientation of the 6-well sheet, and a polycarbonate leg was glued to each end of the plate to raise the plate slightly (-3 mm). To form watertight wells with the 6 holes, 45 x 50 mm glass coverslips (with and without polymer coatings) were mounted over the bottoms of the holes with heat-sterilized high vacuum grease (Dow Corning, Midland, MI).” In control experiments, both fibroblasts and hepatocytes were cultured on TCPS plates with and without vacuum grease applied to the well perimeter: no differences were observed between cells cultured with or without the presence of vacuum grease. Each well in the polycarbonate was notched to accommodate a standard dis- posable sterile petri dish cover. In addition to 6-well plates, individual wells were also constructed from small squares (6 x 6 cm) of 1.2-cm polycarbonate.

Cell culture

Cell maintenance

All cell cultures were maintained at 37°C in a humidified 5% COz atmosphere. 3T3 mouse fibroblasts (BALB/c clone A31, ATCC CCL163, American Type Tissue Collection, Rockville, MD) were passaged prior to reaching confluency and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM with 10% calf serum, 50 pg/mL streptomycin, 50 U/mL penicillin, GIBCO, Grand Island, NY). Chicken embryo fibroblasts (CEF’s) were isolated,’’ maintained in DMEM supplemented as above, and used within two passages. Sus- pensions of 3T3 or CEF cells were produced by detaching the cells from the maintenance flask with trypsin (0.05% with 0.53 mM EDTA, GIBCO) and di- luting to the desired cell density with culture medium. Chicken hepatocytes were isolated by collagenase perfusion” and plated, as described below, within 90 min of collection. Hepatocyte viability in the original suspension was determined by Direct Blue exclusion, and varied from 85 to 93%.

Extent of fibroblast attachment

Glass coverslips or polymer-coated glass coverslips were mounted on the polycarbonate 6-well templates. Six-well TCPS plates (Falcon 3046 35-mm, 6-well plates, Becton Dickinson, Lincoln Park, NJ) were used as additional


controls in some cases. Cell suspension, 2 mL containing approximately lo4 cells/mL, was placed in the chamber directly over each surface. Each culture was incubated overnight (-12 h) at 37°C in a humidified 5% COz atmosphere. At the end of the incubation period the supernatant medium was withdrawn, each chamber was washed three times with sterile Hank's Bal- anced salt solution (GIBCO), and the number of spread cells remaining on the surface was counted by direct observation of at least 10 fields at 1OOX magnification. In these experiments, the area of spread cells was determined by outlining a number of randomly chosen cells on a digital image analysis computer system (Fig. 1).

Fibroblast growth rate

For long-term observations, the cells were maintained in the incubator at 37°C in a humidified 5% COz atmosphere with daily changes of medium. Fibroblasts were maintained in DMEM with 5% calf serum. Coverslips, acid-

Time Lapse Video Tape Recorder

lncu bator

\ \Microscope \ \ \

Cells in Culture \ / on Polymer Substrate \

Figure 1. Computer-based image analysis system for measuring cell speed on surfaces. Surface attached cells in a sealed culture chamber were maintained at a constant temperature for 5 h. During this period, a continuous record of cell movement was produced by the time-lapse videorecorder and serial digital images were collected on the computer.

SALTZMAN ET AL. 746

cleaned or coated with polymer, were mounted in the polycarbonate cell chambers, as above. For each polymer or control surface, eight identically prepared coverslips were mounted in the chambers. All of the cultures were seeded with 2 mL of cell suspension (-lo4 cells/mL) per well. At various times thereafter (9, 48, 96, and 144 h), cell number and viability were determined on two of the polymer substrates. Cell number was determined by counting the number of attached cells observed in 5-10 randomly selected fields of 1 mm’. Cell viability was determined by Direct Blue exclusion. The growth rate was determined by calculating the slope, k, of log(cel1 number) vs. time. The doubling time, t1,2, was calculated from k: tllz = ln(2)/k.

Hepatocyte attachment and growth

Hepatocytes were cultured in Chee’s medium (CEM 5000-7077, B & B Re- search, Hope, RI) supplemented with 100 IU/L insulin (680-3007, GIBCO), 1 pM dexamethasone (D-1756, Sigma Chemical, St. Louis, MO), 9 g/L (3-(N- tris(hydroxymethyl)methylamino)-2-hydroxy-propanesulfonic acid)-sodium salt (TAPSO, T-5395, Sigma Chemical), and 1 ml/L supplemental growth factor (SGF-7, 5000-7668, B & B Research). One milliliter of SGF-7 contains 1 pg epidermal growth factor, 50 p g iron-saturated transferrin, 500 ng selenous acid, 50 p g bovine insulin, 50 pg fetuin, 50 p g oleic acid-BSA, and 50 pg linoleic acid-BSA. Based on the original report,I3 Chee’s medium was modified from Eagle’s essential medium (MEM) to include higher amounts of some amino acids, vitamins, factors such as deoxycitidine and thymidine, mineral salts, and higher buffering capacity. Chee’s medium is superior to Williams’ E and Waymouths media for the maintenance of long-term viability and function of rat hepat~cytes.’~ For our experiments, additional TAPSO was added to the medium to provide increased buffering capacity in 5% COz. Hepatocytes were resuspended in medium containing 200 mg/L gentamicin (600-5750AD, GIBCO) and 2.5 mg/L Fungizone (600-5295AE, GIBCO) and plated (2 mL/well) in wells with polymer and control surfaces. For subsequent medium changes at 9 and 48 h (2 mL/well), the antibiotic concentrations were reduced to 120 mg/L gentamicin and 1.0 mg/L Fungizone.

In addition to the polymer test surfaces described above, hepatocytes were cultured on control surfaces of TCPS 6-well plates, with and without adsorbed collagen (Vitrogen 100, Collagen Corp., Palo Alto, CA). In a laminar flow hood, the highly purified and pathogen-free collagen (approximately 95% collagen I and 5% collagen 111) was diluted to 50 pg/mL with sterile HzO, and then pipetted (1 mL/well) into wells in the TCPS plates. The plates were dried at room temperature in the hood overnight with air flow and without UV irradiation. After drying, the plates were sealed and stored at room temperature for up to two months before use.

Chicken hepatocytes invariably formed large aggregates or f locs in the presence of the adsorbed collagen (see panel labeled collagen in Fig. 9). These aggregates attached only slightly to the collagen surface, so that medium could not be changed in the collagen-adsorbed wells without significant loss


of cells. Therefore the hepatocytes cultured on collagen were maintained in culture with no change of medium for only 48 h.

In preliminary experiments (with 60 mg/L gentamicin and no Fungizone), collagen was air-dried,10~14,’5 l-ethyl-3-(3-dimethylaminopropyl)carbodiimide cross-linked,” and saline-pre~ipitated,“~ onto TCPS plates at 50, 150, and 500 pg collagen/well. As noted p r e v i o ~ s l y , ~ ” ~ ~ ~ ~ ~ and confirmed here, chicken hepatocytes cultured on these surfaces showed no apparent differences in aggregation or attachment during the first 2 days of culture, as detected by microscopic observation at 400X. Finally, rinsed and unrinsed collagen- adsorbed wells were compared. Prior to plating with hepatocytes, wells adsorbed with collagen at 50 pg/mL were incubated with Chee’s medium (without supplements) for 60 min to rinse the wells and rehydrate the collagen. The chicken hepatocytes cultured in the rinsed collagen-adsorbed wells were identical to those in dry, unrinsed collagen-adsorbed wells, as examined by microscopic observation at 400X during the first 2 days of culture.

Chicken hepatocytes were plated at 2 x lo6 viable cells/mL (4 x lo5 cells/ cm’). This cell number results in near confluency and promotes cell viability and f~nction.’~ In previous studies, the mean diameter of chicken hepatocytes was determined to be 13 pm, while rat hepatocytes were determined to be 23 pm.16 The cells in three different wells were counted at each time point: 9,48, 96, and 144 h of culture. In preliminary experiments, it was found that (a) attached and spread hepatocytes were difficult to count, (b) 100% of the attached hepatocytes were viable as indicated by Direct Blue staining, (c) cells were not efficiently detached with trypsin, even after long incubations with full-strength trypsin, and (d) mechanical detachment with a cell scraper tended to shred both the cells and some polymer substrates. Therefore, in subsequent experiments cells were counted with a hemocytometer following detachment from the surface with collagenase (type IV-S, C-5138, Sigma Cherni~al);’~ all detached cells were assumed to be viable.

Collagenase was dissolved at 25 mg/mL in Chee’s medium with 5 mg BSA/mL, aliquoted, and stored at -20°C. To detach the hepatocytes, a well was evacuated of medium, and rinsed with 3 mL of phosphate-buffered saline (PBS). Then, for a 9-h sample, the well was incubated for 12-16 min under ambient conditions with 250 p L collagenase solution and 750 pL Chee’s medium. Substrates with higher concentrations of MVK required longer incubation for efficient cell detachment. For the 48-, 96-, and 144-h samples, a well was incubated for 12-15 min with 100 pL collagenase solution and 900 pL Chee’s medium. Finally, for all samples, whether at 9-, 48-, 96-, or 144- h, the contents of the well were repeat-pipetted approximately 12 times, to ”hose down” the well bottom with the contents of a short glass pasteur pipet. The suspension was then immediately counted in a hemocytometer.

Determination of speed of cell movement

A suspension of CEF’s (1.5 mL) was placed in either a 35-mm TCPS dish (Falcon 1008,35 mm, Becton Dickinson, Plymouth, England) or an individual

SALTZMAN ET AL. 748

polycarbonate chamber with a glass or polymer-coated coverslip. Cells were allowed to attach to the surface for at least 12 h at 37°C in 5% COz, before the medium was changed (DMEM with 5% calf serum and 10 mM HEPES). Im- mediately prior to observation of cell movement, the lid was sealed onto the chamber with sterilized vacuum grease. The sealed chamber was placed on the stage of an inverted light microscope (Nikon Diaphot, see Fig. 1); an air- jacket incubator surrounding the stage was preheated and maintained at 37 & 0.5”C. Using a high numerical aperture 1OX phase contrast objective, a 1-mm2 field of view was randomly selected on the surface. To ensure that the cells and media were not heated during the experiment, an infrared filter was placed in the path of the microscope light beam. Using cell chambers filled with water as controls and monitoring temperature with small copper/ constantan thermocouples (-1 mm3, Omega), we confirmed that water in the focal plane remained at 37°C for 8 h.

A computer-based image analysis system (Compaq 386/20e with Data Translation 2851/2853 image acquisition and analysis boards, Fig. 1) was used to monitor cell movements in the chamber. A video camera (Dage-MTI model no. NC-70) was attached to the microscope and a continuous record of cell movement was produced by a video recorder (JVC model no. BR-9000U) at 1/120th of normal video frame rate. In addition, digitized still images of the field of view were collected at 15-min intervals for 5 h.

After the completion of each experiment, the videotapes and collected images were reviewed simultaneously. For each cell tracked over the course of an experiment, cell position was determined on each stored image by outlining the cell boundary and calculating the center of mass. This procedure produced a record of cell position (x, y ) at discrete intervals over a period of time. Cells were excluded from the analysis if (a) they left the field of view during an experiment, (b) they divided, or (c) they detached from the surface. Cell speed was determined from this record of cell tracks, using the method of Dunn.18,19 This analysis yielded a mean cell speed for the population of cells observed on each surface. Variance was determined by the Jackknife method and 90% confidence limits were obtained from standard T tables. The advan- tages and limitations of this method have been

Staining of the actin cytoskeleton

CEF’s that were allowed to attach to test surfaces overnight were fixed with paraformaldehyde (3.7% v/v) in PBS for 15 min. After a rinse with PBS, the cells were permeabilized with Triton X-100 (0.2% v/v) in PBS for 7 min, and rinsed again with PBS. The triton-insoluble residues were then stained with a solution of 0.06 ,uM rhodamine-conjugated phalloidin (Molecular Probes, Eugene, OR) in PBS for 20 min. After another rinse with PBS, the test surfaces were mounted on microscope slides in a solution containing p - phenylenediamine (0.1% w/v), PBS (10% v/v), glycerol (90% v/v), pH 8.0, and photographed with a fluorescence microscope. To determine the relative area of the spread cells in this experiment, the fluorescent outlines of the cells were traced onto tracing paper. The images of the cells were cut out and weighed.

CELL BEHAVIOR ON POLYMERS

RESULTS

749

All of the polymers tested have good solubility in common organic sol- vents such as methylene chloride and chloroform. The poly(phosphoesters) were biodegradable, but at a slow rate. Previous studies showed that the mass lost from the polymers in the intramuscular space of rabbits ranged from 8% for BPA/PP to 15.2% for BPA/EOP at 11 weeks postimplantation.'* For the cell culture experiments described here, these surfaces can therefore be regarded as nonbiodegradable. These polymers were, however, swellable under in vitro conditions. When tested according to the ASTM method #D570a in 0.1M phosphate-buffered water (pH 7.4), BPA/EOP adsorbed water up to 43 wt% by day 3, while the uptake for the other three poly(phosphoesters) ranged from 4 to 10 wt%. These polymers have fiber- and film-forming properties, as well as adhesiveness to glass and stainless steel. As observed from thermo- gravimetric analysis, the breakdown temperatures ranged from 320°C for BPA/EOP to 510°C for BPA/PP. All four polymers exhibited a Tg around 115°C. The contact angles for the four poly(phosphoesters) were 60" (BPA/EOP), 68" (BPA/POP), 72" (BPA/EP), and 82" (BPA/PP).

Fibroblasts

Fibroblasts attach and spread on all of the surfaces tested; the extent of attachment depends on the chemistry of the polymer surface. Depending on the cell suspension, 70% to 100% of the seeded cells attached to glass or TCPS surfaces. Figure 2 shows the relative attachment of CEF's to poly(styrene- co-methyl vinyl ketone), where the mole percent of styrene in the polymer

Y 100 I

0% 20% 40% 50% 60% 80% 100%

Mole Percent Styrene Figure 2. Relative attachment of CEF's to poly(styrene-co-methyl vinyl ketone). The number of attached cells was determined following 12-h incubation in DMEM with 5% calf serum. Relative attachment was calculated by dividing the number of cells attached to each polymer substrate by the number of cells attached to a control substrate (acid-cleaned glass). For the control substrates under these experimental conditions, between 70% and 100% of the initial cell suspension attached.

750 SALTZMAN ET AL.

140

120 ..

100 .. 80

60

40

20 0 -

was varied from 0% to 100%. In this series of polymers, poly(methy1 vinyl ketone) was the most hydrophilic and the polystyrene the most hydrophobic. For every member of this family of polymers tested, there were fewer cells attached compared to glass coverslips. Maximum cell attachment was seen with the 50% styrene copolymer.

CEF’s also attached to surfaces of the poly(phosphoesters) (Fig. 3). While very few cells attached to the polymer with the ethoxy side chain (the most hydrophilic of the group), cell attachment to some of the polymers (ethyl, phenyl, and phenoxy) was indistinguishable from the glass control. Qualita- tively similar results were obtained with 3T3 fibroblasts (data not shown).

Once the cells attach to a surface, they spread. The distribution of cell area in attached cells followed a log normal distribution, which was characterized by a mean and a standard error (Fig. 4); the mean and standard error for some of the substrates tested are listed in Table 11. The mean projected area of attached CEF’s was approximately 2000 pm2, regardless of the surface. When cell area on glass controls was compared to cell area on every other surface, no statistically significant difference was discovered, suggesting that cell spreading was the same on every surface. Once attached and spread, the cells would proliferate. The doubling time for 3T3 cell growth was approximately the same for all the surfaces tested (Table 111).

Speed of cell movement was estimated by following the tracks of a number of CEF’s migrating on a surface over a 5-h period (Fig. 5). Error in cell speed for a given experiment was estimated by the Jackknife method and is desig- nated on the figure by error bars. The surfaces were divided into two groups: high attachment surfaces on which most (70% to 100% of seeding density) of the suspended cells attached (solid symbols) and low attachment surfaces on which fewer (<50% of seeding density) cells attached (open symbols). In the high-attachment group (glass and TCPS), mean cell speed was in the range 40

.. T _. T -.

N=2 N=2 N=2 N=l N=2

1 . .. ’

Ethoxy Phenyl Ethyl Phenyl Phenoxy (1 0% XL) (6% XL)

Side Chain Group Figure 3. Relative attachment of CEF’s to poly(phosphoesters). The number of attached cells was determined following 12-h incubation in DMEM with 5% calf serum. Relative attachment was calculated by dividing the number of cells attached to each polymer substrate by the number of cells attached to acid-cleaned glass.


V) 0.3 -- - - 8 6 0.2 --

5 0.1 - - E

c 0 .- L

< 500 1000 1500 2000 2500 3000 3500 4000 4500 >5000 to to to to to to to to

1500 2000 2500 3000 3500 4000 4500 5000

Cell Area (pm2 ) Figure 4. Distribution of projected area for cells attached to control and test surfaces. The fraction of cells with projected areas in the indicated range is shown for CEF’s attached to TCPS (filled bars) and BPA/PP/6% (open bars). For the TCPS surface, the mean area was 2100 pm2 with a standard error of 130 pm2 (n = 115). For the poly(phosphoester) surface the mean cell area was 1940 pm2 with a standard error of 150 pm2 (n = 64). This difference in mean cell area was not statistically significant.

TABLE I1 Summary of the Biophysical Studies to Characterize Cell Attachment to

Poly(Styrene-Co-Methyl Vinyl Ketone) Surfaces

Relative Cell Area Attachment Cytoskeletal

Substrate (d) (% control) Speed ( w / h ) Structure

Glass 2100 f 220 Control 4 0 k 6 6% weakispots (n = 92) 4 6 2 8

TCPS 2100 f 130 96 f 28 4 2 f 7 (n = 115) (n = 6) 46-t- 9

N.D.

20% PS 2020 f 220 312 9 67 f 10 25% weakispots (n = 32) (n = 8) 65 f 11

77 k 18

50% PS 1740 2 180 76 k 16 63 * 18 25% weakispots (n = 30) (n = 8) 84 f 16

65 -t- 17

80% PS 2380 f 320 35 f 19 64-t- 8 45% weakispots (n = 28) (n = 6)

Cell areas were determined by tracing cell outlines by computer (mean ? standard error of mean). Cell speeds were determined by the method of Dunn (mean f standard deviation). Cy- toskeletal structure was visualized with rhodamine-labeled phalloidin and scored from blinded photographs (percentage of cells with a weak or diffuse staining pattern or staining in spots). Ab- breviations are defined in Table I. N.D. denotes experiment not done.

752 SALTZMAN ET AL.

TABLE I1 3T3 Cell Growth on Poly(Styrene-Co-Methyl Vinyl Ketone) and Poly(Phosphoesters)

Substrate Doubling Time (h)

TCPS 100% PS 80% PS 60% PS 50% PS 40% PS 20% PS 0% PS BPA/EP BPA/POP BPA/PP BPA/PP/G%

20 20 20 16 28 21 20 30 34 19 22 20

Abbreviations for the BPA polymers are defined in Table I; X% PS indicates poly(styrene-co- methyl vinyl ketone) with X mole % styrene.

to 46 pm/h. The average speed for cell movement on this group of surfaces was calculated by averaging the mean speeds for the four experiments; that average (43 pm/h) is shown by the lower solid line. In the low attachment group (copolymers of styrene and MVK), mean cell speed was in the range 59

20' I I I I I I I I I I I I I I I

Surface Figure 5. Speed of migration of CEF's on polymer substrates. Each point indicates the mean cell speed for a population of cells migrating on a polymer substrate; the number of cells tracked for each determination is indicated. The error bars indicate 90% confidence limits calculated by the Jackknife method. The upper solid line is the average of the experimentally determined mean cell speed on the surfaces indicated by the open symbols; the lower line is the average speed for cells on surfaces indicated by the filled symbols.


to 84 pm/h. The average speed for cell movement on this group of surfaces was calculated by averaging over the 9 experiments; the upper solid line indicates this average (68 pm/h).

Since we employed the method of Dunn to determine mean cell speed, cell persistence time in direction of movement was also determined. Because the period of observation (5 h) was short compared to the persistence time of fibroblasts (-1 h), variance in the estimates of persistence time was high. This same problem was encountered by Dunn in his original description of the method. We found no statistically significant differences in persistence time for cells moving on the different surfaces.

To assess whether this increase in cell speed correlates with alterations in cytoarchitecture, fluorescently labeled phalloidin, which binds specifically to filamentous actin (F-actin):’ was used to stain the actin cytoskeleton. The cells attached to glass coverslips show a normal fibroblast morphology (Fig. 6A-D), with prominent actin filament bundles (stress fibers) running the full length of the cells predominantly at lines of stress. Each bundle consists of dozens of individual actin filaments, which are not resolved at this magnification (1OOX objective). The cells attached to poly(styrene-co-methyl vinyl ketone) with 40% styrene (Fig. 6E-H) show a more diffuse or weak staining pattern, with fewer bundles and some abnormal F-actin aggregates (bright spots). On the polymer substrates tested, 20-4576 of the surface attached cells look similar to the cells in Figure 6E-H; the remaining cells look similar to ones spread on glass (Fig. 6A-D). We found no significant difference in the average area occupied by the fluorescent cells spread on the different surfaces tested, a result consistent with those described in Figure 4.

Figure 61-J show examples of cells attached to poly(styrene-co-methyl vinyl ketone) with 20% styrene. These cells showed unusual swirling/gnarled filament bundles, a pattern which might indicate improper cell attachment and less defined lines of stress. Only a few of the cells on this surface have this morphology; most of the cells look like those in Figure 6A-D or 6E-H. This type of staining pattern was not seen on any other surface.

Figure 6K-L show typical cells attached to a glass surface following ex- posure to a low concentration M) of cytochalasin D, a drug which specifically disrupts actin as~embly.’~ These cells are similar to the cells in Figure 6E-H. Since the formation of actin bundles depends on proper adhesion to the surface, the results of actin staining experiments may indicate that the polymer surface did not provide the proper signal for normal assembly of bundles.

The results of studies with CEF’s on control and test surfaces are summa- rized in Table 11.

Hepatocy tes

Hepatocytes attached, grew, and maintained viability on all the test polymer surfaces and TCPS. The result of one experiment are shown in Figure 7, in which the attachment, growth, and 6-day viability of hepatocytes on poly(styrene-co-methyl vinyl ketone) and TCPS surfaces are compared. The

754 SALTZMAN ET AL.

Figure 6. Photographs of cells stained with rhodamine-labelled phalloidin (magnification SOOX). Individual cells were attached to glass coverslips (A-D) and (K-L), poly(styrene-co-methyl vinyl ketone) with 40 mole % styrene (E-H), poly(styrene-co-methyl vinyl ketone) with 20 mole 7% styrene (I-J). The cells shown in (K) and (I) were treated with M cytochalasin D for 1 h prior to staining with phalloidin. Untreated cells attached to glass show prominent actin bundle formation; cells attached to polymer have weaker staining and staining in spots.

0% 20% 40% 60% 80% 100% TCPS 0 24 48 72 96 120 144

Mole Percent Styrene Time (Hrs)

Figure 7. Hepatocyte attachment to poly(styrene-co-methyl vinyl ketone) substrates with varying mole percent styrene. Percentage of cell seeding indicated is the mean of three samples; standard deviations are indicated by error bars. In the bar graph on the left, cell number as a percentage of cell number in the initial suspension is shown for each polymer substrate at 9, 48, 96, and 144 after initiation of the culture; reproducibility between samples of the same polymer was very good. In the panel on the right, the same data are shown without the error bars to more clearly show the change in cell number over the one week experiment.


hepatocytes showed some growth on all surfaces at 48 h, increasing in number by a factor of 1.5 to 2, and then decreasing to almost identical cell den- sities at 96 and 144 h. In a duplicate experiment, in which the cells were maintained for 96 h, hepatocytes attached, grew, and declined similarly to the first experiment, again showing no large differences among the various polymer surfaces (data not shown). In the cell detachment assays for these two experiments, hepatocytes attached to the 0 and 20% styrene copolymer surfaces resisted collagenase detachment much more strongly than hepatocytes attached to surfaces of 60% to 100% styrene copolymer. A 96-h experiment with poly(phosphoester) surfaces and TCPS control also showed little variation in hepatocyte response to the different polymer surfaces (data not shown).

In the experiments reported here, as well as in all preliminary experiments, the morphology of chicken hepatocytes cultured on TCPS with adsorbed collagen was different from that on all artificial polymers surfaces (Fig. 8). The behavior of chicken heptocytes in the presence of adsorbed collagen was also different from the previously reported monolayer attachment of rat hepatocytes to artificial p01ymers.~,~~-~~ During an initial 9-h culture period with a collagen substrate, most of the chicken hepatocytes formed three- dimensional, spheroidal aggregates or f locs, which appeared to be only slightly anchored to the solid substrate. Some of the chicken hepatocytes also attached to the collagen substrates in clumped monolayers. Techniques have not yet been developed for long-term culturing and analysis of the suspended aggregates. Therefore, the hepatocyte cultures on collagen substrates were not maintained more than 3 days, or quantified with respect to cell number.

Figure 8. Hepatocyte morphology following 48 h of culture on poly(styrene-co-methyl vinyl ketone) surfaces, TCPS, and TCPS adsorbed with collagen.

756

DISCUSSION

SALTZMAN ET AL.

In these studies, we have examined the attachment and subsequent behavior of fibroblasts and hepatocytes on two families of polymeric biomaterials. Fibroblasts and hepatocytes attached, grew, and maintained viability on all the polymer surfaces tested. Because of their chemical versatility, and their demonstrated compatibility with cells of different origin, these polymeric biomaterials may be important for cell culture applications.

More cells attached to glass than to any of the synthetic polymers, suggesting a weaker adhesion between cells and the copolymer surfaces. Among the copolymers, attachment was optimal on the 50% styrene copolymer, a surface with intermediate hydrophobicity. While the extent of spreading among cells attached to any surface was the same, a significant fraction of the spread cells had an abnormal pattern of actin filaments as revealed by staining with fluorescent phalloidin. Increased speed of migration on these surfaces may be related to these differences in cell architecture: the subpopulation of cells without prominent bundles can move faster because rearrangement of the cytoskeleton, which must accompany motility, is fa~ter.’~ While we have observed differences in cell behavior on different polymer surfaces, we recog- nize that our results may reflect differential binding of specific cell adhesion molecules, e.g., proteins like the ones present in the serum we added in experiments with fibroblasts, and will investigate this in future experiments.

The poly(phosphoesters) are extremely versatile: a wide range of physico- chemical properties can be obtained. Since the phosphoester bond is cleav- able under physiological conditions, these substrates are biodegradable. In a previous study, the biodegradation rate was controlled by altering the chemical structure of either the backbone or the side chain.21 For instance, a poly(phosphoester) with a trimethylene group in the backbone and an ethyl side chain slowly dissolved in water, while a bisphenol A backbone was stable in buffer for up to 6 months. Properties of the vinyl polymers were controlled by varying the composition of the copolymers. A common feature shared by these polymers is a reactable side chain. This allows covalent attachment of bioactive components, such as polypeptide fragments or choline groups, to the substrates with subsequent modification of the surface chemistry.

For short-term studies, we used fibroblasts derived from chick embryos. These cells are used routinely in biochemical and biophysical studies. Because these cells were obtained directly from the embryo, cell behavior may change during the adaptation to culture. Therefore, CEF’s were used for only the first two passages. For longer studies involving determination of cell growth rates we used a permanent mouse fibroblast cell line because (a) the cells are readily available, (b) other studies of cell growth on polymer surfaces had been performed on 3T3 cells? and (c) mouse fibroblast cell lines are com- monly used for toxicity e v a l ~ a t i o n . ~ ” ~ ~

Cultured hepatocytes may form the basis of extracorporeal therapies for liver failure.””31 For further development of this technology, it is important to understand the interactions between cultured cells and synthetic polymer


surfaces. In addition, several recent reports have demonstrated the potential of hepatocyte/polymer composites as transplantable internal organ^.^^-^^ We will continue our studies of hepatocyte interactions with synthetic polymers, with the goal of identifying the features of polymer surfaces that promote cell function. With that in mind, these present studies represent a first step in the development of surfaces optimized to support the attachment and function of specific cell types.

We thank Professor Saul Roseman for generously providing the chicken hepatocytes; Dr. Diane Lin for helpful discussions; and Annie Tang, Bholi Datta, Peter Zage, Laura McGinity, Kristine Kieswetter, Laura Balog, and Rovena Sobarzo for technical assistance. This work was supported by Grant #EET-8815629 from the National Science Foundation and by the Whitaker Foundation.

References 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

J. Folkman and A. Moscona, “Role of cell shape in growth control,” Na- ture, 273, 345-349 (1976). A. Ben-Ze‘ev, G. S. Robinson, N. L. R. Bucher, and S.R. Farmer, “Cell- cell and cell-matrix interactions differentially regulate the expression of hepatic and cytoskeletal genes in primary culture of rat hepatocytes,” Proc Natl Acad Sci USA, 85, 2161-2165 (1988). E. D. Hay (ed), Cell Biology of the Extracellular Matrix, Plenum, New York, 1981. T. A. Horbett, and M. B. Schway, ”Hydrophilic-hydrophobic copolymers as cell substrates: effect on 3T3 cell growth rates,” 1. Colloid Inter- face Sci., 104, 28-39 (1985). A.S.G. Curtis, J.V. Forrester, C. McInnes, and F. Lawrie, ’Adhesion of cells to polystyrene surfaces,” J. Cell Biol., 97, 1500-1506 (1983). D. E. Ingber, J. A. Madri, and J. Folkman, ”Endothelial growth factors and extracellular matrix regulate DNA synthesis through modulation of cell and nuclear expansion,” In vitro, 23, 387-394 (1987). R.O. Hynes, “Molecular biology of fibronectin,” Annu. Rev. Cell Biol., 1, 67-90 (1985). C. F. Amstein and P. A. Hartman, ‘Rdaptation of plastic surface for tissue culture by glow discharge,” 1. Clin. Microbiol., 2, 46-54 (1975). N. Li, M. Richards, K. Brandt, and K. Leong, “Poly(phosphate esters) as drug carriers,” Polym. Preprints, 30, 454-455 (1989). J. D. Macklis, R. I. Sidman, and M. D. Shine, ”Cross-linked collagen surface for cell culture that is stable, uniform, and optically superior to conventional surfaces,” In Vitro Cell. Dev. Biol., 21, 189-194 (1985). P. K. Vogt, In Fundamental Techniques in Virology, Habel and Salzman (eds.), Academic Press, New York, 1969, pp. 198-211. M.S. Kuhlenschmidt, E. Schmell, C.W. Slife, T.B. Kuhlenshmidt, F. Sieber, Y.C. Lee, and S. Roseman, “Studies on the intercellular adhesion of rat and chicken hepatocytes,“ J. Biol. Chem., 257, 3157-3164 (1982). D.I. Chee, A.W. Boddie, Jr., J. A. Roth, E.C. Holmes, and D. L. Morton, “Production of melanoma-associated antigen(s) by a defined malig- nant melanoma cell strain grown in chemically defined medium,” Cancer Res., 36, 1503-1509 (1976). H.O. Jauregui, P. N. McMillan, J. Driscoll, and S. Naik, ‘Attachment and long term survival of adult rat hepatocytes in primary monolayer cultures,” In Vitro Cell. Dev. Biol., 22, 13-22 (1986).

758 SALTZMAN ET AL.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

P. L. Iverson, L. M. Parthlow, L. J. Stensaas, and F. Moatamed, “Charac- terization of a variety of standard collagen substrates,” In Vitro Cell. De.? Biol., 17, 540-552 (1981). J. Obrink, M. S. Kuhlenschmidt, and S. Roseman, ”Adhesive specificity of juvenile rat and chicken liver cells and membranes,”Proc. Natl. Acad. Sci. USA, 74, 1077-1081 (1977). 0. Seglen, “Preparation of isolated rat liver cells,” Methods Cell B i d , 13,

G. A. Dunn, ”Characterizing a kinesis response,” Agents Actions Suppl.,

P.C. Wilkinson, J. M. Lackie, J.V. Forrester, and G. A. Dunn, ”Chemoki- netic accumulation of human neutrophils on immune-complex coated substrata,” J. Cell Biol., 99, 1761-1768 (1984). R.T. Tranquillo, 8. E. Farrell, E. S. Fisher, and D. A. Lauffenburger, ‘A stochastic model for chemosensory cell movement: application to neu- trophil and macrophage persistence and orientation,”Math. Biosci., 90,

M. Richards, B.I. Dahiyat, D. M. Arm, P. R. Brown, and K.W. Leong, “Evaluation of polyphosphates and polyphosphonets as degradable biomaterials,” J. Biomed. Mat. Res., to appear. H. Faulstich, S. Zobeley, G. Rinnerthaler, and J.V. Small, “Fluorescent phallotoxins as probes for filamentous actin,“ J. Muscle Res. Cell Motil- ity, 9, 370-383 (1988). M. D. Flanagan, and S. Lin, “Cytochalasins block actin filament elon- gation by binding to high-affinity sites associated with F-actin,” 1. Biol. Chem., 255, 835-838 (1980). N. Sawada, A. Tomomura, C. A. Sattler, G.L. Sattler, H. K. Kleinman, and H.C. Pitot, ”Effects of extracellular matrix components on the growth and differentiation of cultured rat hepatocytes,” In Vitro Cell. Dev. Biol., 23, 267 (1987). N. Sawada, A. Tomomura, C.A. Sattler, G.L. Sattler, H. K. Kleinman, and H. C. Pitot, ”Extracellular matrix componenets influence DNA synthesis of rat hepatocytes in primary culture,” Exp. Cell Res., 167, 458-470 (1986). G. Michalopoulos and H.C. Pitot, “Primary culture of parenchymal liver cells on collagen membranes,” Exp. Cell Xes., 94, 70-78 (1975). I. M. Herman, N. J. Crisona, and T. D. Pollard, ”Relation between cell activity and the distribution of cytoplasmic actin and myosin,” J. Celt

H.J. Johnson, S.J. Northup, P.A. Seagraves, P. J. Garvin, and R.F. Wallin, ”Biocompatibility test procedures for materials evaluation in vitro,” J. Biorned. Muter. Res., 17, 571-586 (1983). Guidelines for Blood-Material Interactions, NIH Publication No. 85- 2185 (1985). H. 0. Jauregui, N. T. Hayner, B.A. Solomon, and P.M. Galletti, ”Hy- brid artificial liver,” In Biocornpatible Polymers, Metals, and Composites, Szycher (ed.), Techomic Publishing, Lancaster, PA, 1981, pp. 907-928. J.C.Y. Dunn, M. L. Yarmush, H.G. Koebe, and R.G. Tompkins, “Hepa- tocyte function and extracellular matrix geometry,” FASE B I., 3,

J.A. Thompson, C.C. Haudenschild, K. D. Anderson, J.M. DiPietro, W. F. Anderson, and T. Maciag, “Heparin-binding growth factor 1 in- duces the formation of organoid neovascular structures in vivo,” Proc. Natl. Acad. Sci. USA, 86, 7928-7932 (1989). J. P. Vacanti, M. A. Morse, W. M. Saltzman, A. J. Domb, A. Perez-Atayde, and R. Langer, ”Selective cell transplantation using bioabsorbable artificial polymers as matrices,” J. Pediatric Surg., 23, 3-9 (1988).

29-83 (1976).

12, 14-33 (1983).

281-303 (1988).

Bid., 90, 84-91 (1981).

174-177 (1989).


34. A . A . Demetriou, J.F. Whiting, D. Feldman, S.M. Levenson, N.R. Chowdhury, A. D. Moscioni, M. Kram, and J. R. Chowdhury, "Replace- ment of liver function in rats by transplantation of microcarrier- attached hepatocytes," Science, 233, 1190-1192 (1986).

Received June 26, 1990 Accepted February 6, 1991

fibroblast and hepatocyte behavior on synthetic polymer surfaces

Documents