NOVEL SYSTEM FOR MULTILAYER GROWTH OF ANCHORAGE-DEPENDENT CELLS

Abstract: Anchorage-dependent cells are grown on a novel substratum which is belived to increase the oxygenation of the cells and reduce the formation of free-radicals. The substratum consists of a perfluorocarbon reservoir which is coated with a layer of protein (e.g., collagen or gelatin) which has been perfluoroalkylated. The perfluoroalkylated protein forms a very stable surface to which anchorage-dependent cells can attach and grow. Using this system, mammalian cell cultures have been grown to densities exceeding 10<7> cells/cm<2>. The increased density is attributed to the formation of multiple cell layers which resemble tissue-like structures.

Description: NOVEL SYSTEM FOR MULTILAYER GROWTH OF ANCHORAGE-DEPENDENT CELLS 1. The Field of the Invention The present invention is related to a system for cultivat¬ ing cells. More specifically, the present invention is related to a novel oxygenation system which supports multilayer growth of anchorage-dependent cells. In one embodiment, this is accom¬ plished by seeding the cells on a perfluoroalkylated pro- tein/perfluorocarbon surface to which anchorage-dependent cells can attach and grow to previously unattainable densities and cell layers. 2. Technical Background Cell cultures are cells from a plant or animal which are grown outside the organism from which they originate. These cells are often grown, for example, in petri dishes under specific environmental conditions. Cell cultures are of great importance because they represent biological "factories" capa- ble of producing large quantities of bioproducts such as growth factors, antibodies and viruses. These products can then be isolated from the cell cultures and used, for example, to treat human disease. In addition, tissue cultures are a potential source of tissues and organs which could be used for transplan- tation into humans. For example, tissue cultured skin cells could potentially be used in skin grafts. Finally, tissue cultures usually comprise cells from only one or a few tissues or organs. Consequently, cell cultures provide scientists a system for studying the properties of individual cell types without the complications of working with the entire organism. In vivo, cells form complex multilayer structures which ultimately form tissues and organs. These cells receive their nutrient and oxygen requirements via the blood in the circula¬ tory system. In addition, in order to form tissues and organs, cells must form contacts with each other and with an extracel¬ lular matrix. Extracellular matrices comprise a complex and variable array of collagens, glycosaminoglycans, proteoglycans, and glycoproteins. Together these cellular products form the basal lamina, bone, and cartilage which give tissues and organs their shape and strength. The contact between anchorage-depen¬ dent cells and the extracellular matrix plays a dramatic role in determining the cells' shape, position, metabolism, differ¬ entiation and growth. Like most cellε in vivo, many cells are anchorage-depen¬ dent; that is, they can metabolize and divide only if they are attached to a surface or substratum. Only cells of the circu¬ latory system (e.g., lymphocytes and red blood cells) grow unattached and suspended in solution in vitro. While many anchorage-dependent cells may grow on glass or plastic surfac¬ eε, these cells often lose their ability to differentiate and respond to hormones. For this reason, glass and plastic tissue culture dishes are often coated with an extracellular matrix such as collagen. Unlike cells in vivo, normal cells in culture do not form significant multilayer εtructureε. Under optimal conditions, for example, epithelial cells grow only one cell layer thick (monolayer) , while fibroblast cells at best grow two or three layers thick. Once the growing surface is confluent with cells, normal cells cease to divide and their number actually begins to decline with time. This phenomenon is referred to as "density-dependent inhibition. " The failure of cells to grow to form multilayered struc¬ tures is a major limitation of current tissue culture tech- niques. Cells growing in monolayers often loose the capacity to perform many essential functions that they perform in their respective tissues and organs. This is primarily due to the fact that in vivo these cells are surrounded by other cells which provide many factors needed for normal function and growth. Thus, current research is hindered by the fact that tissue culture techniques do not accurately mimic in vivo biological activity. It is believed that the inability of cells in culture to form multilayer structures is owed, in part, to lack of oxygen- ation. Oxygen, unlike other nutrients, is only sparingly soluble in aqueous media. Thus, cells several cell layers deep do not receive sufficient oxygen to grow and maintain normal biological activity. Conventionally, two methods have been used to improve oxygenation for cell growth in bioprocesεes: mechanical stirring and bubbling. Cultures have been mechani¬ cally stirred to increase oxygen tranεport at the aqueous/air interface and distribute oxygen uniformly in the culture fluid. This results in shear and other problems with anchorage-depen¬ dent mammalian cells which lack a rigid cell wall and are large (10-100 μ ) and very fragile. Alεo, anchorage-dependent mamma¬ lian cells cannot rotate or translate freely to reduce the net forces and torqueε from shear because they are attached and fixed to the substratum. Studies εuggest that the viability of endothelial and human kidney cells is profoundly affected by shear. These effects are observed at εhear rates as low as 1 dyne/cm 2 . Cell shear stress is also created when air bubbles contact the cell or bioparticle. Typically, higher bubbling flow rates per unit volume increase the specific death rate of anchorage-dependent mammalian cells in microcarrier syεtems. Shear stresε may alεo cauεe changes in the εhape and function of cellε. If the εtress is strong enough, anchorage- dependent cells can be detached from the surface to which they are attached. In addition, some cell functions, such as cyto- skeleton assembly, metabolism, biomolecular synthesis, are also shear stress dependent, even under conditions of laminar flow. Many inveεtigators have added a polymer to the medium to reduce hydrodynamic effectε. This has been found to provide some protection. The most frequently used polymers are methyl¬ cellulose, polyεucroεe, Dextran, and Pluronic F-68. It is believed that the polymers adhere to the cell εurface and form a protective εhell against shear and mechanical forceε. Howev¬ er, whether a protective shell is actually formed or the ef- fects it may have on normal cell function is unknown. Another method for increasing oxygenation to cell cultures is the hollow fiber membrane bioreactor. In this system, the cells are attached to a cylindrical hollow fiber membrane. Culture media and oxygen flows through the center of the cylin- drical hollow fiber membrane. The molecular weight cut-off of the membrane permits nutrients and oxygen to reach the cells without allowing the cells to escape. However, the formation of multilayer of cells was not observed. Apart from the problem of poor oxygenation, current tissue culture techniques produce free radicals. Free radicals are atoms or polyatomic molecules which poεεes one unpaired elec¬ tron. In culture media, free radicals arise from the reaction of oxygen with iron, or copper, as well aε certain metaboliteε which are normally preεent in growth media. Studieε have εhown that free radicalε are toxic to mammalian cellε, even at very low levelε. Thus, benefitε derived from increaεed oxygenation of culture media uεing molecular oxygen, would likely be coun- tered by increaεed free radicals. More recently, perfluorocarbons (PFCs) have been used to increaεe oxygenation of cultureε. Perfluorocarbons are organic compoundε where all hydrogen atomε are replaced by fluorine atomε. Oxygen is 15 to 20 times more soluble in PFCs that in water. Aε a result, PFCs are sometimes referred to as "psuedo- erythrocytes" because they are oxygen-carrying molecules analo¬ gous to the erythrocytes that carry oxygen in mammalian blood. Indeed, PFCs have been used as oxygen carriers in place of red blood cells in animals. Current tissue culture techniques which employ PFCs, however, have their own limitations. One method teaches con¬ tinuously adding an oxygenated PFC to the top of the culture media. The PFC being denser than the aqueous culture media sinks to the bottom of the bioreactor where it is removed. While this system succeεsfully improved the oxygenation of the culture media, it has two significant disadvantages. First, the εystem iε limited to suspension cells which must be mechan¬ ically stirred in order to prevent them from settling at the bottom of the reactor. As diεcussed above, this damages ma ma- lian cells. Second, the PFC comeε into direct contact with the cells. PFC has been shown to alter the normal biological activity of various cells in culture. For example, neutrophils and monocytes incubated with PFCs exhibit decreased phagocytic activity, chemotaxis, aggregation, cellular adherence, and superoxide ion release. Currently, cultivation of anchorage-dependent mammalian cells using PFCs has been unεucceεεful. The principal diffi¬ culty is that anchorage-dependent mammalian cells do not adhere to PFC surfaces or do not adhere any better than on conven¬ tional polystyrene surfaceε. A few studies have reported some growth on microspheres (diameter 100 to 500 μm) made by emulsi¬ fying perfluorotertiary amine. It was found, however, that the cells were actually growing on a layer of protein desorbed from the serum used in the nutrient medium. This layer was not εtable and growth waε erratic. Moreover, the cultures had to be vigorouεly εtirred to enεure equilibration with oxygen resulting in cellular damage. Finally, since serum contains very little extracellular matrix material, the microspheres did not provide a good subεtratum for anchorage-dependent cell growth. In fact, growth on theεe microspheres was not as good as on commercially available microcarriers fabricated with collagen, or gelatin. From the foregoing, it will be appreciated that it would be an advancement in the art to provide a system which provideε improved oxygenation to anchorage-dependent tissue culture cells. It would be a further advancement in the art if the an- chorage-dependent tissue were able to form multilayer tisεue- like εtructureε. It would be yet another advancement in the art if the improved oxygenation could be accomplished without mechanically stirring or agitating the culture media. It would also be an advancement in the art if the increased oxygenation reεulted from perfluorocarbon moleculeε that were not in direct contact with the tisεue culture cellε. Finally, it would be an advancement in the art if the amount of free radicalε produced in the culture media were greatly reduced or eliminated. Such a εyεtem iε diεclosed and claimed herein. 4. BRIEF SUMMARY OF THE INVENTION The present invention is directed to a novel oxygenation system which supports multilayer growth of anchorage-dependent cells. More particularly, the invention relates to a substratum for growing culture cells in vitro which is capable of forming 3-dimensional tissue-like structures. The substratum consists of a reservoir of oxygenated perfluorocarbon (PFC) . The sur¬ face of the PFC reservoir is coated with a layer of an extra¬ cellular matrix protein, or other protein to which culture cells can attach, which has been perfluoroalkylated. Per- fluoroalkylation chemically bonds a PFC tail to the extracellu¬ lar matrix protein. The PFC tail of the protein can then bind with the PFC reservoir, anchoring the protein and creating a strong, stable surface on which anchorage-dependent culture cellε can attach and grow. Thiε novel tiεεue culture technique overcomeε several problems facing the art. First, the perfluoroalkylated pro- tein/perfluorocarbon (PF-protein/PFC) subεtratum of the present invention is capable of continually supplying high concentra¬ tions of oxygen to the culture cells. The high affinity of PFC for oxygen permits high concentrations of oxygen to pass through the PF-protein interface and reach the culture cells. No mechanical stirring or agitation is required. Depending on the intended use, the PFC reservoir can either be a closed system with a finite supply of oxygen or an open system which is continuously regenerated with oxygen. In one preferred embodiment, HeLa cells were seeded on perfluoroalkylated gelatin (PF-gelatin) which waε anchored to an open εystem perfluorodecalin (PFD) reservoir. After 15 days, the cells had reached a density of 1.1 x 10 7 cells/cm 2 . More importantly, a 3 micron vertical section stained with hematoxylin and eosin εhowed that cellε had grown to form more than 19 layerε. Mitotic figureε deep within the multilayer demonstrate that the cells were receiving enough oxygen in order to divide. Second, the system of the present invention limits free radicals. As discussed above, free radicals are toxic to mammalian cells at even very low concentrations. PFCs do not contain free radicals. Moreover, the PFC which delivers oxygen to the cells never comes in direct contact with the growth media. The PFC and the aqueous growth media are separated by the PF-protein interface and at least one layer of cells. Thus, the oxygen is not as available for reaction with the iron, copper and other metaboliteε in the growth media which produce free radicals. Finally, the PFC iε never in direct contact with cells. Aε diεcussed above, PFCs have been shown to adversely affect cells in culture. In the syεtem of the preεent invention, the PFC reεervoir iε εeparated from the cellε by the PF-protein interface which the cells grow on. Thus, the harmful effects of PFCε on cellε iε greatly reduced. Taken together, the increaεed oxygenation, reduced free radicalε, and reduced exposure to PFCs, haε reεulted in a novel system for growing cells which yields three dimensional tisεue- like εtructureε never before achieved in culture. Theεe and other objectiveε and advantages of the present invention will become apparent by examination of the following description of the accompanying drawingε, the detailed description of the invention and the appended claims. 5. SUMMARY OF THE DRAWINGS A more particular description of the invention briefly described above will be rendered by reference to the appended drawings and graphε. Theεe drawingε and graphs only provide information concerning typical embodiments of the invention and are not therefore to be considered limiting of itε scope. Figure 1 is a croεε sectional view of one embodiment of the present invention illustrating cells grown on a perfluoro- alkylated-gelatin/perfluorodecalin subεtratum in a test tube. Figure IA is an expanded view of the perfluoroalkylated- gelatin/perfluorodecalin - cell interface of Figure la. Figure 2 is a graph illustrating HeLa cell growth (cells/cm 2 ) on different εubstratumε. Black circleε represent growth on perfluorodecalin treated with perfluoroalkylated gelatin. Black squareε repreεent growth on perfluorodecalin treated with gelatin that had not been perfluoroalkylated. Black diamondε represent growth on untreated perfluorodecalin. Open diamonds represent growth on a conventional polystyrene surface. Figure 3 is a graph illustrating HeLa cell growth (cells/cm 2 ) in test tubes containing varying amounts of per- fluorodecalin. Figure 4 is a croεs sectional view of one embodiment of the present invention illustrating a U-εhaped teεt tube uεed to culture HeLa cells. On one neck of the tube, HeLa cells are grown on a perfluoroalkylated-gelatin/perfluorodecalin substra¬ tum, while the opposite neck contains no cells and permits limited reoxygenation of the perfluorodecalin reservoir. Figure 5 is a graph illustrating HeLa cell growth (cells/cm 2 ) in test tubes containing varying amounts of per- fluorodecalin versus HeLa cell growth in a U-shape test tube which permits some reoxygenation of the perfluorodecalin reser¬ voir. Figure 6 is a photograph of a hematoxylin and eosin stained 3 micron vertical thin section of a 15-day old HeLa cell culture grown on a perfluoroalkylated-gelatin/perfluoro¬ decalin subεtratum in the U-εhaped teεt tube configuration. The dark εtained areas are the nuclei. Thiε photograph waε taken with a green filter at 40x magnification. 6. DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to a novel oxygenation system which supports growth of anchorage-dependent cells. This iε achieved using a substratum capable of supporting 3-dimen¬ sional tissue-like structureε. Reference is now made to the figures wherein like parts are referred to by like numerals. With reference to Figure 1, in one preferred embodiment, the present invention is a system 10 for multilayer growth of anchorage-dependent cells 20. The system 10 comprises a test tube 12, a perfluorocarbon (PFC) reservoir 14, a PF-protein/PFC substratum 16 and aqueous growth media 22. The system is constructed by first adding an appropriate amount of oxygenated PFC to the bottom of a sterile test tube 12 or other container, thus forming a PFC-reservoir 14. On the surface of the PFC reservoir 14 a PF-protein/PFC substratum 16 is formed. Aqueous growth media 22 is then layered over the PF-protein/PFC εub- stratum 16. Finally, anchorage-dependent cells 20 are added to the growth media 22 where they settle to the PF-protein/PFC subεtratum 16, attach and grow. With reference to Figure IA, the PF-protein/PFC εubstratum 16 compriseε a protein 18, εuch aε gelatin or other protein to which culture cellε can attach, which has been perfluoro¬ alkylated. Perfluoroalkylation substitutes one of the hydrogens of a free amino group of the protein 18 with a PFC tail 17. With continued reference to Figure IA, the PFC tail 17 of the protein 18 binds with the PFC reεervoir 14, anchoring the protein 18 and creating a strong stable surface. Once the PFC tail 17 binds to the PFC reservoir 14, conventional aqueous growth media 22 is supplied and anchorage-dependent culture cells 20 are seeded on the PF-protein/PFC subεtratum 16. The anchorage-dependent culture cells 20 are finally overlaid with conventional aqueous growth media 22. Thus, the oxygen re¬ quired for cell viability and growth is supplied both by the PFC reservoir 14 which is underneath the anchorage-dependent culture cells 20 and the aqueous growth media 22 which is above the cells. Nutrients and growth factors are supplied by the aqueous growth media 22 which is above the anchorage-dependent culture cells 20. To better understand the detailε of the invention, the discusεion of the invention below iε divided into three parts: the perfluorocarbon (PFC) reservoir, the PFC-protein/PFC sub- εtratum and cell growth. 5.1 PERFLUOROCARBON RESERVOIR In the present invention, the PFC reservoir deliverε high concentrationε of oxygen to the tiεεue culture cellε. Per- fluorocarbonε are organic compoundε where all hydrogen atomε are replaced by fluorine atomε. The carbon-fluorine bond of perfluorocarbons is so strong (116 kcal/mol, compared with C-H, 80.36 kcal/mol) that they are very stable and inert for biolog¬ ical processes. Importantly, PFCs do not produce free radi- cals. Perfluorocarbons are also quite apolar compounds. Since the solubility of gaseε increases as the polarity of the εol¬ vent decreases, PFCs are useful gas transferring molecules. For example, oxygen is 15 to 20 times more soluble in PFC than water. As a result, PFCs are of great interest in tisεue culture. Other properties of PFC molecules are well documented in patents and scientific articles, including King, A. T. , B. J. Mulligan and K. C. Lows, "Perfluorochemicals and Cell Cul¬ ture," Bio/Technol . , 7(10), 1037-1042 (1989) and Faithful, N. S. , "Second Generation Fluorocarbons," Adv . Exp . Med . Biol . 317: 441-52 (1992) which are hereby incorporated by reference. The structure of some PFCs is shown below: (a) Perfluorotributylamine (b) Perf luorobutyltetrahydrof uran (c) Perfluorodecalin (PFD) Other PFCs which would be useful in the present invention include fluorinated liquids manufactured by Minnesota Mining and Manufacturing Company (3M) . It is understood by one skilled in the art that this list is not exhaustive but merely representative of the types of PFC molecules that fall within the scope of this invention. 5.2 PF-protein/PFC Substratum 5.2.1 Protein Moεt mammalian cellε are anchorage-dependent. Therefore, attachment to a compatible εubεtratum is neceεεary for growth. Thiε approximateε in vivo environmental conditionε where cellε grow as a continuum, attached to other cells or to basal mem¬ branes by the extracellular matrix (ECM) . A special protein (called transmembrane linker protein) binds to a cell surface receptor (called intracellular attachment protein) and then interacts with the extracellular matrix so that the cell atta- ches to the matrix. One of the most important compounds in ECMs is collagen. Collagens are abundant in mammals, constituting about 25 weight percentage of the total protein. As many aε 15 different types of collagens have been identified. The first four types, labeled I, II, III and IV, have been uεed for attachment and growth. Typeε I, II and III are the moεt abundant and form fibrils and the linear structure. Type IV is a two-dimenεional εheetlike structure and forms the two-dimensional reticulum in ECM. It is well known and documented in the prior art that different types of cells adhere to certain types of collagen. Gelatins are denatured collagens. The natural conforma¬ tion of the collagen is stabilized by several types of interac¬ tions, principally hydrogen bonding. This conformation can be destroyed and denatured by heating or by alkaline treatment. Heating collagens, generally at 60-65°C, causeε the helices to unravel. On cooling the triple-helical conformations are regenerated to some extent. However, these reconfirmations are not exactly like the original collagens and are called gela¬ tins. Anchorage-dependent cells also adhere well to surfaces treated with gelatin. The present invention is not limited to collagen or gelatin. Other important proteins in the extracellular matrix to which cells attach are expected to fall within the scope of the present invention. These include, but are not limited to, fibronectins, laminins and elastins. Fibronectinε and lamininε are εubstrate adhesion molecules (SAM) which help cells bind to the extracellular matrix. Elaεtinε are cross-linked random coils, inεoluble proteins that give tissue its elasticity, and are the main components of elastic fiberε. 5.2.2 Perf luoroalkylation Overlaying an extracellular protein directly on the PFC reεervoir would not provide a εtable enough εurface for the attachment and growth of anchorage-dependent cellε. Therefore, in one preferred embodiment the protein is perfluoroalkylated. The technique involveε uεing a perfluoroalkylating agent to chemically bind a PFC to a protein. This provides a hydropho- bic perfluorocarbon tail which sinkε into the PFC reservoir surface, thus firmly anchoring the protein to the PFC reser¬ voir. The general perfluoroalkylation reaction is: PF-NC=0 + R-NH + PF-NCO-NH-R where PF-NC=0 iε the perfluoroalkylating agent and R-NH + repre¬ sentε an amino group in the protein. Because the isocyanate group reacts with water, the reaction must be carried out in a moisture-free environment. In one preferred embodiment, gela- tin was perfluoroalkylated with perfluorooctylpropyl isocyanate (C 8 F π (CH 2 ) 3 NCO) . However, other perfluoroalkylating agents with alkyl groups of varying lengths or ring structureε could be used to perfluoroalkylate proteins. After the reaction, the efficiency of the reaction is determined using a fluorescamine aεεay. Briefly, the per¬ fluoroalkylated protein iε reacted with fluorescamine which readily reacts with free amino groups. The fluorescence of the molecule is then measured at 350 nm. The efficiency of per- fluoroalkylation varies with different proteins. This iε due to a difference in number and acceεεibility of the amino groupε. For example, gelatin iε perfluoroalkylated poorly compared to bovine serum albumin (BSA) . Therefore, the effi¬ ciency of perfluoroalkylation should be determined for each protein used to insure that the protein is adequately per¬ fluoroalkylated. 5.2.3 PF-protein/PFC Interface Once the desired protein is perfluoroalkylated, it must be bonded to the surface of the PFC reservoir. Enough PF-protein is added to each test tube to completely cover the top surface of the PFC reservoir. Aε illuεtrated in Figure IA, the PFC tail of the PF-protein sinks into the PFC reservoir. The teεt tubeε are allowed to stand at room temperature for at least 12 hours to allow complete bonding of the PF-protein to the PFC reservoir and to allow the PFC to equilibrate with air. Excesε PF-protein εolution is then gently aspirated off. Tensiometric measurementε were made to determine the εtability of the PF-protein at the PFC/aqueouε interface. In one preferred embodiment, a du Nouy ring uεed to measure inter¬ facial tension was immersed in a PFD reservoir. The PFD was then overlaid with PF-gelatin. After the PF-gelatin had bonded and equilibrated, water was slowly layered over the PF-gelatin. The resultε εhow that the interfacial tension at the PFD/PF- gelatin interface was approximately 32 dyne/cm, while the interfacial tension of PFD/water measured approximately 50 dynes/cm. The significantly lower interfacial tension indi¬ cates that the PF-gelatin bonded uniformly across the PFC surface. 6.3 CELL GROWTH 6.4.1 Culture Conditions and Growth on Different Substratums In one preferred embodiment, once the PF-protein was bonded and equilibrated with the PFC reservoir, approximately 1-2 x 10 s cells/cm 2 were εeeded on the surface of the PF-pro¬ tein/PFC interface using techniques well known in the art. In one preferred embodiment, a suεpenεion of HeLa cells was tryp- sinized from a confluent stock culture and εeeded at an initial cell denεity of 1.8x10 s cellε/cm 2 in Dulbecco's Modified Eagle'ε Medium (DMEM) εupplemented with 10% Bovine Serum, glutamine (350 mg/liter) , pyruvate (110 mg/liter) , 30nM N-2-Hydroxyethyl- piperiazine-N-2'-2-ethane sulfonic acid (HEPES), penicillin (100 Units/ml) , streptomycin (lOOμg/ l) and 33 mM NaHCθ 3 . The cells quickly settled to the surface and adhered to the PF- protein surface. The tubes were incubated at 37.5°C in an air- 5% C0 2 incubator. They were replenished with fresh medium every 2 dayε during the first 3 to 5 days of culture, and more fre¬ quently after that to keep the pH between 7.2-7.4. For compar¬ ison, HeLa cells were also grown on PFD without gelatin, on PFD overlaid with gelatin that had not been perfluoroalkylated, and on conventional polystyrene tissue culture plates. A comparison of the growth rates on these different sub- stratums is illustrated in Figure 2. It will be appreciated that after a variable delay of 1-2 days (lag phase) , the cellε on the PF-gelatin εurfaceε grew at an exponential rate until day 8. Exponential rates of growth indicated that the cells in the culture were healthy and undergoing mitotic division. After this exponential phase, the growth acceleration declined although the overall cell density continued to increase for at least 17 days, reaching a final density of about 9 x 10 6 cells/cm 2 . In comparison, cells did not grow at all when plated directly on PFD. In fact, the number had declined after 4 days and few cells survived. Cells inoculated on PFD covered only with nonperfluoroalkylated gelatin attained an exponential growth rate that was much lower than the PF-gelatin system. Again, inspection of the cultures indicated that most of the cellε were aggregating around the rim of the teεt tube, proba¬ bly adhering to and growing on the gelatin which had migrated to the wallε of the polystyrene tubes. The final cell density in tubes with nonperfluoroalkylated gelatin was only about 4.8 xlO 5 cells/cm 2 . These data demonstrate that perfluoroalkylation of the gelatin is essential for cell growth. Finally, Figure 2 reveals the superiority of culturing cells on the substratum of the present invention over conventional polystyrene plates. Cells grown on polystyrene plates grew at log phase only until day 6. Moreover, cell denεity only reached a maximum of ap¬ proximately 2.49 xlO 6 cellε/cm 2 . It will be appreciated by one εkilled in the art that numerous other cell types may be grown on the PF-protein/PFC substratum of the present invention. Examples include, hepato¬ cytes, liver cells, cancer cellε, nerve cellε, heart cellε. In addition, it will be appreciated that the present invention is particularly well suited for growing co-cultures which is hereby defined aε εimultaneouεly culturing multiple cell typeε. Finally, it will also be appreciated that in accordance with the prior art, the specific growth media, extracellular protein and incubation conditions may be altered to meet the needs of the particular cells being cultured. 6.4.2 Varying Volumes of PFC Based on the premise that density-dependent inhibition results in part from inadequate oxygenation, decreaεing the volume of the PFC reservoir should result in a concomitant decrease in cell density. To test this theory, in one preferred embodiment, a series of test tubeε were εet up with 0.25, 0.3 and 0.88 mis PFD. Since 0.3 mis of PFD did not cover the bottom of the tube, small glasε beadε (diameter 50-100 μm) were added to provide the same top surface area as in tubeε with 0.88 mlε. PFD haε a very low surface tension so that the 0.3 mlε permeat¬ ed between the beads leaving a thin layer on top on which the PF-gelatin was bonded. A slightly smaller volume (0.25 mis) was also tested uεing specially designed flat bottom test tubes which were made to have the same cross sectional area. It is seen in Figure 3 that in tubes with 0.88 mis of PFD, cells continued to grow for 10 days reaching a final cell density of 8.8 x 10 6 cellε/cm 2 aε in the previouε experimentε. In contraεt, in the tubeε with only 0.3 mlε and 0.25 mlε PFD, both round and flat bottom, growth stopped at 10 days at a density of only 2.2 x 10 6 cells/cm 2 . Thiε finding εuggeεts that the high cell densitieε reached uεing PF-gel/PFD εubεtratumε were not due to the εtate of the collagen. They are most rea¬ sonable ascribed to better oxygenation at the substratum due to diffusion from PFD. The findings also suggeεt that cellε stopped growing in the polystyrene wellε aε a reεult of oxygen depletion at the polystyrene interface. 6.4. Open vs . Closed Reservoir PFC System In order to study the effect of reoxygenation of the PFC reεervoir, cell cultures grown in teεt tubeε (closed syεtem) were compared to cellε cultured in a teεt tube which permitted the PFC reservoir to reoxygenate (open syεtem) . With reference to Figure 4, in one preferred embodiment, cellε were cultured in a U-εhaped teεt tube 30. The PFC reεervoir 14 waε formed by adding 9 mlε of PFD to the U-shaped test tube 30. To the surface of the PFC reεervoir 14 (on only one arm of the U- εhaped test tube 30) was bonded a PFC-protein/PFC substratum 16 to which anchorage-dependent cellε 20 could attach and grow. Finally, both arms of the U-shaped test tube 30 were filled with aqueous culture media 22. In all other respects both sideε of the U-tube were treated identically. With continued reference to Figure 4, in order not to diεturb the cellε during feeding, a tuning fork shaped pipet 32 was used to add and remove liquid from both sideε of the U-shaped tube 30 εimulta- neously. The results of culturing the cells in an opened versus a closed system are illustrated in Figure 5. The growth rates for both curves are about the same. However, the exponential phase of growth in the U-tube lasted about 24 hourε longer than the exponential growth of cellε in the teεt tubes. Cells in the U-tube also were maintained at a higher cell density than thoεe in the teεt tubeε, reaching a denεity of 1.0 x IO 7 cellε/cm 2 at day 17. The more prolonged exponential growth phaεe and the higher cell denεity that could be maintained are even more εignificant in view of the fact that the U-tube contained only 5 mlε of growth medium whereaε the teεt tubeε had 11 mlε. Also, the pH in the U-tube declined so rapidly that after 6 days they had to be replenished with growth medium every 5 hours. Even with this frequent replenishment, the pH could not be maintained above 7.2 which is below the optimal pH for HeLa growth. 6.4.4 Multilayer Formation As diεcussed above, HeLa cells growing in U-tubes on a PF- gelatin/PFD substratum attained densities of about 1.0 x IO 7 cells/cm 2 . In contrast, cells grown in conventional systems using polystyrene wells reached densities of only about 2.6 x IO 6 cells/cm 2 . This suggeεted that with improved oxygenation cells were able to grow in layers on top of each other instead of being reεtricted to the "monolayer" obtained on εolid εur¬ faces. To test this theory, a 15-day old culture from a U-tube was fixed, embedded, εtained and sectioned using histological techniques well known in the art. Briefly, the cells were fixed with glutaraldehyde and dehydrated by soaking them in increasing concentrations of ethanol and finally in acetone. Fixed and dehydrated cells were then slowly raised to the top of the U-tube and embedded in glycol methacrylate and benzoyl peroxide. The block of embedded tissue was sectioned and stained with hematoxylin and eosin which stain the nuclei and the cytoplasms of the cells blue and pink, respectively. A photomicrograph of a thin 3 micron vertical section of the embedded culture is shown in Figure 6. Cells grew to form a tissue-like structure of more than 19 layers. It iε also important that the bottom side of the layers adhering to the PF-gelatin is parallel with the upper surface. This strongly indicates that the architecture was indeed due to cells growing on top of each other to form many layers and not just due to a superficial aggregation of the cells. The section is also free of any necrotic areas aε seen in spheroids. Most importantly, mitotic figureε are obεerved deep in the εection indicating that the level of oxygen needed for growth waε maintained across the multilayers of cells. 7. EXAMPLES The following exampleε are given to illustrate various embodiments which have been made with the present invention. It is to be understood that the following examples are not comprehensive or exhaustive of the many types of embodiments which can be prepared in accordance with the present invention. Example 1 Purification of Gelatin: In one experiment, the protein uεed to construct the PF- protein/PFC substratum of the present invention was gelatin. Before use, the gelatin was purified aε followε. Commercial gelatin (Sigma, G-2500, Type A, 300 Bloom) was fractionated using 50% v/v of isopropyl alcohol (IPA, EM Sciences, PX 1835- 1) . All equipment used was autoclaved and all εolutionε ster¬ ilized by filtration through a sterilized 0.2 μm filter. Two autoclaved bottles were used to εtore 60 mlε IPA and 60 mis 50% IPA diluted with deionized water. Both bottles were stored in a freezer. Gelatin (10 mg/mlε ) waε diεεolved in 50 mlε of water by εtirring at room temperature. Fifty milliliterε of cold IPA were added to the 50 mlε of the gelatin εolution. Thiε bottle was stored in the freezer for at least 24 hours. The precipitate was collected by centrifugation at 4000 G at 5°C for 15 minuteε and the εupernatant removed. Fifty millili¬ ters of cold 50% IPA solution were added with gentle mixing and stored in the freezer for at least 24 hours and the solution was again centrifuged and the supernatant discarded. The washed precipitated gelatin was resuεpended in 10 mlε of 0.1 M εodium phosphate buffer solution, pH 8.5, and stored at 4°C. Example 2 Preparation and Asεav of Perfluoroalkylated Gelatin: The purified gelatin from Example 1 was then perfluoro¬ alkylated. The perfluoroalkylating agent (PF-agent) , perfluoro¬ octylpropyl isocyanate (C 8 F 17 (CH 2 ) 3 NC0) , was donated by du Pont Co. The isocyanate group reactε with water and therefore, the perfluoroalkylation waε done in a moiεture free atmosphere. A glove box was flushed with nitrogen which had been passed through a canister of Drierite (W. A. Hammond Drierite Co.), and then through a 0.2 μm filter to ensure a moisture free sterile atmosphere. Ten microiiters of the PF-agent were added to 40 microiiters HPLC-grade acetonitrile (Aldrich, 27071-7) and vortexed for 5 εecondε. Any unused PF-agent solution was returned immediately to storage. The 50 microiiters PF- agent/acetonitrile mixture was then added to 9 mis of purified gelatin and stirred for 2 hourε. The number of amino groups of the gelatin that were per¬ fluoroalkylated was determined by a fluorescamine assay. Fluorescamine conjugates readily with free amino groups. A calibration curve was made uεing glycine. A εtandard solution of 80 μg/mls glycine (Sigma, G-7126) was prepared in a 0.1 M sodium phoεphate buffer εolution, pH 8.5. A stock solution of fluorescamine (0.3 mg/ml ) waε made in OmniSolv acetone (EM Science, AX0116-3) . Assay tubes were set up for calibrating a range of glycine concentrations from 0 to 8 μg/ml. After adding the fluorescamine, the tubes were vortexed and allowed to stand for 10 minuteε. The fluorescence was measured uεing a Model A-4 System Filter Fluorometer (Optical Technology Devic- es, Inc.) at an excitation wavelength of 350 nm. The concen¬ tration of amino groups versus fluorescence was fit by linear regresεion. The gelatin εolutions were assayed in the same way. The fluoreεcence value obtained on an assay of the native gelatin indicated the total number of amino groups available for per- fluoroalkylation. The fluorescence of PF-gelatin assayed at the same concentration indicated the relative number of amino groups which had been perfluoroalkylated. The fraction of reacted groups is calculated as follows: v=n _ one. of amino groups in PF-gelatin Cone, of amino groups in gelatin where y is the ratio of reacted amino groups in PF-gelatin and cone, is the concentration of amino groupε obtained from the calibration curve. Example 3 Bonding of the Perfluoroalkylated Gelatin to PFD: The perfluoroalkylated gelatin of Example 2 waε then bonded to the PFC reεervoir as followε. Seven εterile 15 mis tissue culture polystyrene test tubes (Falcon, 3033) were used for each experiment. Perfluorodecalin (0.88 ml) was added to each test tube. This was sufficient to cover the round bottom of the test tube providing a cross sectional surface area of 1.33 cm 2 as a subεtratum for growth. PF-gelatin (0.5 ml) waε added to each tube to completely cover the top surface of the PFD reservoir. The tubes were allowed to stand at room temper¬ ature for at leaεt 12 hours to allow complete bonding of PF- gelatin to the PFD reservoir and to allow the PFD to equili¬ brate with air. Excess PF-gelatin solution was then gently aspirated off. Example 4 Tensiometer Experiment: Once the PF-protein had bonded to the PFC reservoir, tensiometric measurements were made to determine the stability of the PF-protein at the aqueous/PFC interface. Twenty milli¬ liters PFD were added to an empty beaker. A du Noiiy ring, a platinum ring used to measure interfacial tenεion, was immersed completely the PFD. Five milliliters of perfluoroalkylated gelatin (10 mg/mls ) were then added and the two phases were allowed to equilibrate for 12 hours. Next, 10 mis of water were slowly added. The interfacial tension of the PFD/aqueous and aqueous/air interfaces were measured at certain time intervals after water being adding. The results indicate that the PF- gelatin/PFD interfacial tension was about 32 dyne/cm, while the PFD/water interfacial tenεion waε about 50 dyne/cm. The lower interfacial tenεion at the PF-gel/PFD interface indicates that a very stable surface had formed. By contrast, the interfacial tenεion of the water/PF-Gelatin interface approached the inter¬ facial tension for water/air surfaces. Example 5 Culturincf HeLa Cells on PF-qelatin/PFD Substratum: The PF-protein/PFC subεtratum of Example 3 waε then seeded with anchorage-dependent HeLa cells. Specifically, a suspen- sion of HeLa cells was trypsinized from a confluent stock culture and diluted into DMEM containing 33 mM bicarbonate and 10% bovine calf serum to give about 2.2X10 4 cells/mis. After flushing with carbon dioxide, 11 mis were added to PF-gel/PFD containing test tubes (diameter 1.33 cm 2 ) giving an initial cell density of 1.8x10 s cells/cm 2 . Cells quickly settled to the surface and adhered to the PF-gel/PFD subεtratum. The tubeε were incubated at 37.5°C in an air-5% C0 2 incubator. They were replenished with fresh medium every 2 days during the first 3 to 5 days of culture, and more frequently after that to keep the pH between 7.2-7.4. One tube was withdrawn after 2, 4, 6, 8, 10, 13, and 17 dayε of incubation. The medium waε removed. The cell layer rinsed with PBS and then trypsinized for cell counts. Growth was expresεed aε an increaεe in cell denεity, e.g., cellε/cm 2 aε determined by cell countε. Example 6 Culture Conditions: HeLa cellε of Example 5 were cultured by methods well known in the art. Briefly, HeLa cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) (Sigma Chemical Co. #D-7777) containing 10%. Bovine calf serum (Hyclone Laboratorieε # A- 2111) , 1% Penicillin-εtreptomycin from a 5000 units/mis peni- cillin and 5 mg/mls streptomycin εtock (Sigma Chemical Co. #P- 0906) diεsolved in εterile DMEM, 2.5% of a 1 M solution of N-2- Hydroxyethylpiperazine-N-2'-2-ethanesulfonic acid (HEPES), and 3.3 % of a 1 M solution of sodium bicarbonate. Cultures were incubated at 37.5±0.2°C in an air-5% C0 2 incubator. Cell pre- pared from a frozen stock were passed at least once before being uεed in experimentε. Example 7 Cells Form Multilayers in the PF-Gelatin/PFD Syεtem: Cellε were grown aε deεcribed in Example 6 except that a U-tube (Fiεher, 09-235A, 1.3 cm diameter and about 1.33 cm 2 εurface area) containing 9 mlε of PFD waε uεed. After 15-dayε in culture, the cellε were fixed, embedded, εtained and sec¬ tioned as followε. The medium in the U-tube waε removed uεing the double barreled pipette and the U-tube waε rinsed with phosphate buffered saline to remove nonadhering cells. The layer was fixed by two 10-minute treatmentε with glutaralde¬ hyde. This stabilizes cell structure and prevents autolysis. The cells were then dehydrated for 20 minute each in 70%, 80% and 95% ethanol and finally 100% acetone. The fixed and dehydrated layer of cells was then slowly raised to the top of the U-tube so that it could be treated for embedding. The cell layer was then removed εlowly uεing a syringe pump (Orion Research Inc. , Model 355) to pump PFD into the other arm of the tube that did not have cellε. The PFD waε pumped in at a rate of 3 mls/min. After the layer was raised to the top, it was floated off into a small vesεel where it could be embedded. Thiε waε done in two stepε. Briefly, 5 mlε of Solution A containing glycol methacrylate (GMA) monomer waε added. The preparation waε allowed to εtand 2 hourε in order for the monomer to infiltrate into the cell layers. Thiε procedure waε repeated by removing old Solution A and adding freεh Solution A. Then Solution A waε removed and an embedding εolution waε added for about an hour. This solution consiεted of a mixture of 5 mlε Solution A and 0.4 ml Solution B containing benzoyl peroxide, which cata- lyzeε the polymerization of the monomers and hardens the layer. The block of embedded tisεue waε sectioned and stained with hematoxylin and eosin. Hematoxylin was used to stain the nucleus of a cell blue and eosin was used to stain the cyto¬ plasm pink. A photomicrograph of a thin 3 micron vertical section of the embedded culture is shown in Figure 6. 8. SUMMARY In summary, growth of cells on PF-protein/PFC substratumε can εupport εustained growth of cells in a three-dimensional tissue-like structure. Growth in multilayers is believed to be primarily due to three factors. First, high concentration of oxygen is delivered to the cells by PFCs. Second, the oxygen is delivered to the cells from a PFC reservoir beneath the cells and also from the aqueouε medium above the cellε. This minimizes the amount of oxygen coming from the aqueous media which contains metalε and metaboliteε which produce free radi- calε when reacted with molecular oxygen. Finally, the cellε are not in direct contact with the PFC which has been shown to affect the biological activity of some cells in culture.

Claims: CLAIMS : 1. A method for the attachment and growth of cells comprising the steps of: a. contacting a surface with a perfluorocarbon; b. bonding a perfluoroalkylated protein to the per¬ fluorocarbon such that a perfluoroalkylated/per- fluorocarbon substratum is formed; c. adding aqueous growth media; and d. contacting the perfluoroalkylated/perfluorocarbon substratum with cells. 2. The method of attaching and growing cells of claim 1 wherein the perfluorocarbon iε εelected from the group conεist- ing of perfluorodecalin, perfluorotributylamine and perfluoro- butyltetrahydrofuran. 3. The method of attaching and growing cellε of claim 1 wherein the perfluoroalkylated protein haε a perfluorocarbon tail, and wherein the perfluorocarbon tail iε perfluorooctyl¬ propyl. 4. The method of attaching and growing cells of claim 1 wherein the perfluoroalkylated protein is selected from the group consisting of gelatins, collagens, fibronectins, lamininε and elastinε. 5. The method of attaching and growing cells of claim 1 wherein the cellε are eukaryotic cells. 6. The method of attaching and growing cells of claim 5 wherein the cells are co-cultured. 7. The method of attaching and growing cellε of claim 1 wherein the eukaryotic cellε are εelected from the group con¬ sisting of HeLa cells, hepatocytes, liver cells, cancer cells, bone marrow cells, nerve cells, heart cells and co-cultures of the above. 8. The method of attaching and growing cellε of claim 6 wherein the cellε grow to form multiple cell layerε following contact with the perfluoroalkylated/perfluorocarbon substratum. 9. The method of attaching and growing cells of claim 1 wherein the aqueouε growth media iε not in direct contact with the perfluorocarbon. 10. The method of attaching and growing cells of claim 1 wherein the εurface contacted with perfluorocarbon is a cloεed εystem. 11. The method of attaching and growing cells of claim 1 wherein the εurface contacted with perfluorocarbon iε an open εyεtem. 12. A method for the attachment and growth of cellε comprising the steps of: a. contacting a surface with a perfluorocarbon; b. perfluoroalkylating a protein; c. bonding the perfluoroalkylated protein to the per¬ fluorocarbon such that a perfluoroalkylated/per- fluorocarbon substratum is formed; d. contacting the substratum with aqueouε growth media; and e. contacting the perfluoroalkylated/perfluorocarbon substratum with at least one anchorage-dependent eukaryotic cells and allowing the anchorage-dependent eukaryotic cells to grow. 13. The method of attaching and growing cells of claim 12 wherein the perfluorocarbon iε εelected from the group conεist- ing of perfluorodecalin, perfluorotributyIamine and perfluoro- butyltetrahydrofuran. 14. The method of attaching and growing cells of claim 12 wherein the protein is selected from the group consisting of gelatins, collagenε, fibronectinε, lamininε and elastins. 15. The method of attaching and growing cellε of claim 12 wherein the eukaryotic cellε are selected from the group con¬ sisting of HeLa cellε, hepatocyteε, liver cellε, cancer cellε, nerve cellε, heart cells and co-cultures of the above. 16. The method of attaching and growing cells of claim 12 wherein the cells form multiple cell layers. 17. The method of attaching and growing cells of claim 12 wherein the aqueous growth media is not in direct contact with the perfluorocarbon. 18. The method of attaching and growing cellε of claim 12 wherein the surface contacted with perfluorocarbon is a closed system. 19. The method of attaching and growing cellε of claim 12 wherein the surface contacted with perfluorocarbon is an open system. 20. A surface for the attachment and growth of cells compris- ing: a perfluoroalkylated protein having a perfluorocarbon tail; and a perfluorocarbon; wherein the perfluorocarbon tail of the perfluoro- alkylated protein bonds with the perfluorocarbon, forming a stable surface on which anchorage-dependent cells can attach and grow. 21. The surface for the attachment and growth of cells of claim 20 wherein the perfluorocarbon is selected from the group consisting of perfluorodecalin, perfluorotributylamine and perfluorobutyltetrahydrof ran. 22. The surface for the attachment and growth of cells of claim 20 wherein the protein is selected from the group con¬ sisting of gelatins, collagenε, fibronectinε, lamininε and elaεtins. 23. The surface for the attachment and growth of cells of claim 20 wherein the perfluorocarbon tail is perfluorooctyl¬ propyl isocyanate.

Inventors: RAPPAPORT CATHERINE, TRUJILLO EDWARD M, RAPPAPORT, CATHERINE, TRUJILLO, EDWARD, M.
Applicants: UNIV UTAH RES FOUND, UNIVERSITY OF UTAH RESEARCH FOUNDATION
Application year: 1996
Main classification: C12M
IPC codes: C12M3, C12N5