Morphological Differentiation of Colon Carcfnoma Cell Lines in Rotating Wall Vessels

Microgravity has advantages for the cultivation of tissues with high fidelity; however, tissue formation requirescellular recognition and adhesion. We tested the hypothesis that simulated microgravity does not affect celladhesion. Human colorectal carcinoma cells were cultured in the NASA Rotating Wall Vessel @WV) under lowshear stress with randomization of the gravity vector that simuIates microgravity. After 6 7 days, cells wereassayed for binding to various substrates and compared to cells grown in standard tissue culture flasks and staticsuspension cultures. The RWV cultures bound as well to basement membrane proteins and to CEA,anintercellular adhesion molecule, as control cultures did Thus, microgravity does not alter epithelial cell adhesionand may be useful for tissue engineering.INTRODUCEON-Production of complex tissues requires that individual cells recognize and adhere to &er; cells and to extracellularmatrix and basement membrane molecules. Microgravity may be useful in engineering new tissues in vimbecause the lack of sedimentation may permit the formation of tissues with high fidelity to their in vivocounterparts. However, microgravity must not alter the ability of cells to recognize and adhere to extracellularmatrices and intercellular ligands if it is to be used to construct tissues. The purpose of this study was to test thehypothesis that microgravity does not affect cell adhesion. This was tested in the NASA Rotating Wall Vessel(RWV) in which cells suspended in aqueous medium rotate around the horizontal axis under conditions of lowshear stress. The rotation randomizes the gravity vector and simulates some aspects of microgravity. Humancolorecral carcinoma cells were used as a model for epithelial cell adhesion because they use the same adhesionreceptors as normal epithelium. Cells were cultured for short (6 7 days) or long (23 days) periods and assayed-.. for binding to standard adhesion molecules. Although the amount of CD44 was decreased in long term cultures,.:. cells grown in simulated microgravity had the same binding characteristics as did cells grown in standard tissue' culture flasks. Thus, microgravity may be useful for tissue engineering..r:MErHODOLcGY-a. L!?kuhxThe EFT-29KM cell line is a sub-line of the ELT-29 cell line /I/. The cell line KM-12c was established at the U.T.M.D. Anderson Cancer Center/2,3/. MIP-101 was established by Kdes et aL 141, while CCL 188 was obtainedfrom the-American Type Culture Collection (Rockville, MD). All cells were grown and maintained at 37OC in ahurnidifie'd'atmosphere containing 5% C02 HT-29Kh/l, CCL 188, and KM-12c were maintained in Dulbecco'sminim& essential media (DMEM) supplemented with 10% fetal calf serum, and 0.30 mg/ml of L-glutamhe. ME-101 cells were grown in RPMI-1640 media supplemented with 7.5% fetal calf serum, and 0.30 mg/ml of L-glutamine. All cell lines were cultured in the presence of 100 unitslml of penicillin and 100 pg/ml ofstreptomycin. Medium was changed every third day and monolayers of cells (85-90% confluent) were harvestedby mild nypsinization. After harvesting, cells were counted using a hemocytometer and viability determined bynypan blue dye exclusion. Cells were routinely 85-90% viable. All culture media and sera were obtained from Sigma Chemical Co., S t Louis MO. Cultures were routinely tested for the presence of mycoplasma by stainingwith Hoechst dye 33258 and were negative.Monolaver Cells were seeded into T25 flasks (Costar) or nonadherent bacterial Petri dishes (Falcon) at 3 x 105 ceus/ml intheir standard culnue medium. Cells were cultured for the indicated number of days, harvested by qpsinization,and then tested in the adhesion assay.Rotating-Wall Vessel fRW culnnqThe RWV is a 125 ml vessel that rotates around the horizontal axis. Cells were added to dextran micmarierscoated with collagen (Cytodex-3 beads) (Phmacia, Piscataway, NJ) at 2 x 105 celldml (or a total of 5.0 x 107cells), which resulted in a starting concentration of 10 cells per bead since the concentration of beads was 5mg/ml. After inoculating the RWV, the cultures were allowed to grow for 48 hours before the medium waschanged. Thereafter, the medium was changed every 20 to 24 hours; as the metabolic requirements of thecultures increased, fresh medium was supplemented with an additional 10 to 200 mgldl of glucose. Cells wereharvested for the adhesion assays by dissociation with collagenase and DNase.# Adhesion AssayIQuantitation of the binding between nunor cells and the various substrates was determined by a modification of themethod described by Hostetter et al. /5/. The substrates were dried, at a concentration of 1 pg per well in. -phosphate buffered saline (PBS), onto 96 well plates (Costar, Cambridge, MA) for 24 hours at 31°c and theplates washed with PBS. The free binding sites on the plastic were blocked with 4% BSA in PBS for 2 hours at37OC and the plates washed with PBS. The cells were prelabelled with 300 pCi 5 1 ~ r(Na2Cr04, specific activity450 mCi/mg, New England Nuclear, Boston, MA) for I hr at 37OC The cells were washed with calcium-freePBS, harvested by mild trypsinization and plated at concenbaions of lx ld t cells per well in the coated plates.After cenaifugation (30g x 5 mins), the plates were incubated for two hours at 37OC. Nonadherent cells wereremoved by three washes with 0.5% BSA in PBS and the remaining cells were solubilized with 1M NaOH andradioactivity collected on cotton-tipped swabs. These were counted for 51Cr using a Tracor Analytical gammacounter. The percent of cells bound was deiined as the ratio of the mean number of counts in an experimental welldivided by the mean number of counts plated in each well. The concentration of 1 pglwell of CEA and other-substrates was chosen from titration studies with KM-12c as well as repetitive experiments which indicated thatoptimal and reproducible binding was achieved at that concentration.? RESULTSGrowth in the RWV Compared to Standard Tissue Culture Svstem~Human colorectal carcinoma cells in the RWV routinely achieved cell concentrations of0.5 1.5 x 106 ceWml or. greater in short term cultures of up to 10 days figure 1A). When cells were cultured for longer periods (20 30days), they grew to greater concentrations before plateauing m-29KM to 4 5 x 106 cells/ml/l/ or MIP-101cells to 5 x 106 ceWml at 23 days). These cell densities are greater than the 0.9 2.0 x 106 ceWml achievedwith these cell lines in standard monolayer cultures in tissue culture flasks.a.... Epithelial Cell Adhesion Under .MiaogravityGrowth of Human Colorectal Carcinomas in the RWV )ICCL 1885;5x= 103-d6 00510DAYFig. 1 Cells were added to Cytodex 3 microcarrier beads and cultured in the RWV as described in the"Methodology."Adhesion to Laminin and Collagen in Standard Culture Svs teqCells cultured in standard monolayer cultures were assessed for their ability to bind to proteins in extracellularmatrix and basement membranes as well as to QEA, an intercellular adhesion molecule. CCL 188 and KM-12c. .cells bind to laminin and CEA while HT-29KM and MIP-101 cells bind to laminin alone (Table 1). None of the. . . .. ... colorectal carcinoma cell lines bind to fibronectin.TABLE I Adhesion of Human Colorectal Carcinoma Cells Cultured in Standard Tissue CultureFlasks To Molecules Attached To a Solid Phase.. CEA% Cells Bound (Mean + SEM)CCell Line Production None BSA Fibronectin Laminin CEAHT-29KiM 300 1 k . l 1 2 .2 2 t . l 34 + 1 1 2 1CCL 188 97 1 t .1 2 2 .3 l + . l 43 C 4 16 + 1KM1 2 ~ 1950 3 t 2 3 + 1 1 2 C 7 6 0 k 7 2 1 + 3_MIP-101 0 1 0 2 1 9 + 1 11+ 1 80 rf: 6 10+1Bold print represents a significant difference from the negative controls of EcO.001 by Scheffe testCEA Production is in ng/ml of medium.When the effect of time in culture was examined, there was a relative decrease in the adhesion of cells to collagenor laminin up to 3 days after plating in monolayer cultures. Both KM-12c 2) and IET-29KM cells (Figure3) had a relative decrease in the number of cells that bind to collagen and hminh but both cell lines hadsignificantly greater binding to collagen and laminin than they did to CEA, fibronectin, or the control protein BSA(Figures 2 and 3). Similarly, when cells were plated on bacterial Pem dishes so that they remain in suspensionbut do not attach to a surface, KM-12c cells maintained their ability to bind to collagen and laminin (Figure2);however, HT-29KM lost its ability to bind to collagen and laminin within three days (Figure 3). Both cell linessustained a large loss of viability when cultured in static suspensions for more than three days that precluded anyfurther evaluation. Interestingly, the adhesion to CEA by KM-12c ceb was not great in either the monolayer orstatic suspension culture system (Figure 2). This may be because the expression of CEA by recently plated cellsis low and only increases as cells attain the plateau phase of growth. Nonetheless, cell adhesion in RWV cultureswere compared to adhesion in standard monolayer cultures because the static mspihion cultures were largely, nonviable. (Q74I. M. Iestup er al.Fig. 2A.2B.KM-12c Cells In Standard Monolayer CultureKM-12c Cells in Static Suspension Culture 01230123DayDayFig. 2I Fig. 3A.3B.HT-29KM Cells In Standard Monolayer CultureHT-29KM Ceils in Static Suspension Culture 01230123-DayDayFig. 3-