WO1994003586A9 - A method for detaching intact cells and reducing implant rejection - Google Patents
A method for detaching intact cells and reducing implant rejectionInfo
- Publication number
- WO1994003586A9 WO1994003586A9 PCT/US1993/007371 US9307371W WO9403586A9 WO 1994003586 A9 WO1994003586 A9 WO 1994003586A9 US 9307371 W US9307371 W US 9307371W WO 9403586 A9 WO9403586 A9 WO 9403586A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- plasminogen
- cells
- monolayer
- recipient
- substrate
- Prior art date
Links
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Definitions
- the present invention provides a means for obtaining a high yield of intact cells. This invention is particularly useful where there is a limited source of such cells.
- the method of the present invention separates a monolayer of cells from its substrate (as a sheet or a suspension of separate cells) without damaging the cells.
- the method of the present invention is applicable to medicine (for example for covering a tissue transplant or a prostheses with autogenous cells) , immunology, gene engineering and biotechnology.
- proteolytic enzymes such as trypsin, collagenase, pronase
- chelating agents see S. oskalewski, G. Thyberg, Cell Tissue Res., v. 220, p.p. 51-60, (1981); W. Halle, M. Melzig, W. Schosler, W. E. Siemg, W. Viola, E. Teuscher, Biomed. Biochem. Acta, v. 45, N 10, p.p. 1315-1324, (1986) ; M. A. Gimbron, R. S. Cotrau, J. Folkman, Cell Biol., v. 60, p.p. 673-684 (1974), namely, that proteolytic enzymes attack cell membrane surface proteins, which attack apparently disrupts the cellular membrane and produces "deficient" cells.
- proteolytic enzymes such as trypsin, collagenase, pronase
- chelating agents see S. oskalewski, G. Thyberg,
- trypsinized dog aorta endothelial cells contained anomalous giant cells. This cell culture degenerated after five subcultivations.
- the method for cell subcultivation of the present invention has the following advantages:
- the detaching process of the present invention increases (or generates) plasmin activity around the to be separated cell.
- plasmin activity hydrolyses adhesive proteins that connect cells to their substrate.
- the plasmin activity is increased (generated) by adding plasminogen to the medium.
- the plasminogen activator may be evolved in the media during cell growth.
- the evolved plasminogen activator is adsorbed onto the cells.
- the to be separated cells are incubated in a serum-free medium with an increased (generated) plasmin activity for: at least about 30 minutes at a temperature in the range of about 18°C and about 45°C; at least about six hours at a temperature in the range of about 4°C and about 15°C; or a combination thereof.
- the working solution of plasminogen is stable (i.e., does not lose its activity) under normal storage conditions.
- the material to be inserted into a patient e . g. , a dog, cat, horse, human
- a specimen of viable cells is obtained from the patient. These cells are grown to a sufficient number and then either (i) inserted into the patient directly as described, for example, by C.C. Compton et al .
- the material-cell combination is inserted into the patient.
- the number of cells is sufficient to bind, on average, 10 3 cells per mm 2 of the material to be inserted into the patient.
- the process of the present invention enables one to prepare a culture of the cells with out transforming any cells.
- plasminogen is used to separate the cells from their substrate, transformed cells are not introduced into the patient the cells are inserted.
- Fig. 1 shows a monolayer of BHK-21 cells grown in a plastic flask (Greiner, 50 ml) under standard conditions
- Fig. 2 shows a monolayer of BHK-21 cells grown in a plastic flask (Greiner, 50 ml) under standard conditions, 8 subcultivations after trypsin treatment;
- Fig. 3 shows degenerated BHK-21 cells, 12 subcultivations after trypsin treatment
- Fig. 4 shows an SDS- electrophoresis gel of dog plasminogen preparation used to detach a cell monolayer from its substrate and molecular weight markers
- Fig. 5 shows mono!aver of BHK-21 cells grown in a plastic flask (Greiner, 50 ml) , after 19 subcultivations where the monolayers were separated by incubation for 37°C in a fresh serum-free media containing dog plasminogen for 5 hours followed by a 15 hour incubation in cold conditions, +4°C to +6°C;
- Fig. 5A shows an SDS-electrophoresis gel of (1) human, (2) bovine plasminogen and (3) dog, as well as (4) a standard mixture of protein molecular weight markers.
- Fig. 6 shows a roll of BHK-21 cells after monolayer detachment from the substrate by incubating the monolayer with dog plasminogen at +4°C to +6°C; and
- Fig. 7 shows a cell suspension obtained by pipetting a plasminogen detached monolayer of BHK-21 cells.
- Plasminogen was prepared from the dog serum by a modification of the affinity chromatography method of D.G. Deutch and E.T. Mertz (Science, v.170, p.p.1095-1096 (1970)) which is hereby incorporated by reference. The purity of plasminogen preparations was estimated by SDS- electrophoresis as described by U.K. Laemmly (Nature, v.??7, p.p. 680-685 (1970)), which is hereby incorporated by reference.
- Plasminogen was identified by its N-end amino acid sequence by an automatic Edman degradation (about 30 residues) .
- the partial primary structure of dog plasminogen is reported by Y. Schaller, C. Straub, U. Stammer, E.E. Rickli, Complete Amino Acid Sequence Of Canine "Miniplasmino ⁇ en” , 2 Protein Seq. Data Anal. 445- 50 (1989) .
- bovine plasminogen The primary structure of bovine plasminogen is reported in J. Schaller, P.W. Moser, G.A.K. Dannegger- Muller, S.J. Rosselet, U. Courter and S.E. Rickli, Complete amino acid sequence of bovine plasminogen (comparison with human plasminogen) , Eur. J. Biochem., V.149,. p.p. 267-278 (1985). These references on the primary structure of plasminogens are hereby incorporated by reference.
- plasminogen was sterilized by filtering it through an 0.22 mm membrane filter.
- the buffer had a 1 mg/ml plasminogen protein concentration.
- the volume of plasminogen solution we added was between about 0.01 to about 0.1 volumes of the fresh medium.
- Our dog plasminogen working solutions had a plasminogen concentration, in the media, in the range of about 10 to about 50 ⁇ g/ml. We found that the amount of plasminogen necessary depends on the method of cell separation.
- bovine and human plasminogens were substituted for dog plasminogen using the conditions described herein. However, as described in detail below, we used more concentrated working solutions of the bovine and human plasminogens.
- DMEM Dulbecco's Minimum Eagle's Medium
- the mammalian plasminogens at some conditions (a temperature, a period of incubation, a plasminogen concentration) detach cells from their substrates.
- the detached cells are viable, spread rapidly, and grow well.
- the dog plasminogen was more active than either bovine or human plasminogen.
- Plasmin hydrolyses adhesive proteins which are believed to connect cells to the substrate. Some enzymes convert plasminogen into plasmin, two of which may be found in a cell culture: namely, tissue-type and urokinase-type plasminogen activators. Cells can adsorb them from the serum during a cell grow. Many cultured cells have receptors for either or both enzymes. Plasminogen activators may bind to cellular receptors and localize plasmin formation. (See D.P. Beebe, Thromb. Res., v. 46, p.p. 241-254 (1987); K.A. Hajjar, N. M. Hamel, P. C.
- Initial mammalian plasminogens concentrations for detaching cells from their substrate are about the same as the concentrations of the plasminogen in the blood in the species from which the plasminogen is isolated.
- Plasminogen or plasmin appears to act with high specificity on adhesive molecules without damaging cell membranes. Plasminogen detached cells can undergo prolonged passaging of cells, preserving their typical morphology (and presumably, their cell membranes) .
- plasminogen or plasmin detaches cells, and cells detached this way undergo significantly more cell passages (the upper limit was not tested) than cells detached using commercial trypsin solution.
- Example l protocols used BHK-21 cells that were cultured in 50 ml plastic flasks (Greiner company) under standard conditions (DMEM with 10% bovine serum) .
- DMEM fetal calf serum
- plasminogen solution 1 mg/ml
- a saline buffer typically, a 0.01 M Na 2 HP0 4 , pH 8.3 saline buffer
- the medium had a 0.075 mg/ml plasminogen concentration.
- EXAMPLE 4 - (Prophetic Dog Prostheses Example) A sample of endothelial cells from a dog is surgically removed and aseptically transferred to an appropriate primary explanation cell culture medium. See, e.g., J. Paul, CELL & TISSUE CULTURE, which is hereby incorporated by reference. The cells are grown under appropriate aseptic conditions through several subcultivations. After several subcultivations, a dog blood-vessel prostheses is placed in the flask and used as a growth substrate. After a monolayer of endothelial cells has formed on the prostheses, the prostheses is surgically implanted in the dog from which the endothelial cells had been removed.
- a monolayer of endothelial cells from a primary dog explanation cell culture is carefully detached from a growing flask and introduced between the threads of a blood-vessel prostheses. The cells are grown on the prostheses until they substantially cover the prostheses' surface. At that time, the prostheses is surgically implanted in the dog from which the endothelial cells had been removed.
- EXAMPLE 5 - (Prophetic Human Prostheses)
Abstract
The application describes a means of detaching cells from their substrate without damaging or transforming the cells. The cells detached from their substrate by the methods of this application can be used to prepare a tissue implant or to prepare exogenous cells to coat a prothesis to reduce the risk of rejection.
Description
A METHOD FOR DETACHING INTACT CELLS AND REDUCING IMPLANT REJECTION Background
The present invention provides a means for obtaining a high yield of intact cells. This invention is particularly useful where there is a limited source of such cells. The method of the present invention separates a monolayer of cells from its substrate (as a sheet or a suspension of separate cells) without damaging the cells. The method of the present invention is applicable to medicine (for example for covering a tissue transplant or a prostheses with autogenous cells) , immunology, gene engineering and biotechnology.
Conventional methods for separating the cells from their substrate (namely, treatment with trypsin, collagenase, pronase or a similar enzyme) have important limitations. For example: a) Cell cultures, when detached using conventional methods, frequently stop growing after several subcultivations. b) Conventional methods transform some cells. Such transformations may be responsible for cancers
(after a cell or tissue transplant, clinicians have reported results that suggest this possibility, see M. S. Borzy, R. Hong,
S. D. Horowitz, E. Gilbert, D. Kaufman, . De Mendonea, V. Oxelius, M. Dictor, L. Pachman, N. Engl. J. Med. , v. 301, p.p. 565-568 (1979) .
We have discovered an important disadvantage of
conventional methods results from cell subcultiva ion with proteolytic enzymes (such as trypsin, collagenase, pronase) and/or of chelating agents (see S. oskalewski, G. Thyberg, Cell Tissue Res., v. 220, p.p. 51-60, (1981); W. Halle, M. Melzig, W. Schosler, W. E. Siemg, W. Viola, E. Teuscher, Biomed. Biochem. Acta, v. 45, N 10, p.p. 1315-1324, (1986) ; M. A. Gimbron, R. S. Cotrau, J. Folkman, Cell Biol., v. 60, p.p. 673-684 (1974), namely, that proteolytic enzymes attack cell membrane surface proteins, which attack apparently disrupts the cellular membrane and produces "deficient" cells.
Trypsinized fibroblasts from Syria hamster cell line, BHK-21, undergo morphological changes and do not form a continuous monolayer by the eighth subcultivation, (Fig. 1.2) . After twelve subcultivations, the trypsinized BHK-21 cell culture degenerated (Fig. 3) .
After four subcultivations, trypsinized dog aorta endothelial cells contained anomalous giant cells. This cell culture degenerated after five subcultivations. The method for cell subcultivation of the present invention has the following advantages:
(a) One can obtain cells with intact cytoplasmatic membranes and free of destructive nucleus modifications.
(b) The viability of primary cell cultures is increased. This increase makes practical cultures from a limited source by obtaining the highest possible yield of cells (for example, to cover a tissue transplant, or a protheses, with autogenous cells or to prepare a vaccine from the cells of an exotic animal) . (c) Few, if any, cells are transformed. Avoiding transformation is essential if the cell culture is to be introduced into an organism, say as a coating on a tissue transplant or a protheses. Brief Description of the Invention The process of the present invention separates cells from other cells and substrates without viable harm to the cells being separated. Instead of using harsh
proteolytic enzymes such as trypsin, collagenase and pronase and/or chelating agents, the detaching process of the present invention increases (or generates) plasmin activity around the to be separated cell. (Plasmin hydrolyses adhesive proteins that connect cells to their substrate) . In a preferred embodiment, the plasmin activity is increased (generated) by adding plasminogen to the medium.
As some cells synthesize and secrete plasminogen activators, the plasminogen activator may be evolved in the media during cell growth. The evolved plasminogen activator is adsorbed onto the cells.
Typically, the to be separated cells are incubated in a serum-free medium with an increased (generated) plasmin activity for: at least about 30 minutes at a temperature in the range of about 18°C and about 45°C; at least about six hours at a temperature in the range of about 4°C and about 15°C; or a combination thereof.
It is preferred that the to be separated cells are separated using a media that is substantially free of proteolytic enzymes other than plasmin (i.e., substantially free of trypsin, collagenase and pronase as these proteolytic enzymes are not added to the serum media in preferred embodiments of the present invention) . Material that increases (produces) the plasmin activity can be prepared in advance in a readily used form. For example, plasminogen can be dissolved/dispersed in a carrier such as a saline buffer at a concentration sufficient for use of the plasminogen in carrier as a working solution for addition of plasminogen to a cell media to detach the cells therein. It is preferred that the working solution of plasminogen is stable (i.e., does not lose its activity) under normal storage conditions. In one embodiment of the method of reducing rejection of a material inserted into an organism (for example, a tissue transplant or a protheses) of the
present invention, the material to be inserted into a patient ( e . g. , a dog, cat, horse, human) is coated with cells from the patient before the material is inserted. Thus, in a first step, a specimen of viable cells (such as epithelial and endothelial cells) is obtained from the patient. These cells are grown to a sufficient number and then either (i) inserted into the patient directly as described, for example, by C.C. Compton et al . , 60 Laboratory Investigation p.p. 600-12 (1989) , which is hereby incorporated by reference, or (ii) bound to the material to be inserted into the patient. Thereafter, the material-cell combination is inserted into the patient. Typically, it is desirable that the number of cells is sufficient to bind, on average, 103 cells per mm2 of the material to be inserted into the patient.
Moreover, the process of the present invention enables one to prepare a culture of the cells with out transforming any cells. Thus, when plasminogen is used to separate the cells from their substrate, transformed cells are not introduced into the patient the cells are inserted. Brief Description of the Figures
Fig. 1 shows a monolayer of BHK-21 cells grown in a plastic flask (Greiner, 50 ml) under standard conditions; Fig. 2 shows a monolayer of BHK-21 cells grown in a plastic flask (Greiner, 50 ml) under standard conditions, 8 subcultivations after trypsin treatment;
Fig. 3 shows degenerated BHK-21 cells, 12 subcultivations after trypsin treatment; Fig. 4 shows an SDS- electrophoresis gel of dog plasminogen preparation used to detach a cell monolayer from its substrate and molecular weight markers;
Fig. 5 shows mono!aver of BHK-21 cells grown in a plastic flask (Greiner, 50 ml) , after 19 subcultivations where the monolayers were separated by incubation for 37°C in a fresh serum-free media containing dog plasminogen for 5 hours followed by a 15 hour incubation
in cold conditions, +4°C to +6°C;
Fig. 5A shows an SDS-electrophoresis gel of (1) human, (2) bovine plasminogen and (3) dog, as well as (4) a standard mixture of protein molecular weight markers. Fig. 6 shows a roll of BHK-21 cells after monolayer detachment from the substrate by incubating the monolayer with dog plasminogen at +4°C to +6°C; and
Fig. 7 shows a cell suspension obtained by pipetting a plasminogen detached monolayer of BHK-21 cells. DETAILED DESCRIPTION OF THE INVENTION
In the course of culturing dog aorta endothelial cells, we observed that after adding dog blood serum (10% v/v) to the culture medium, the monolayer of cells separated from the substrate after a 12 to 24 hour cold incubation (+4°C to +6°C) (USSR Author's certificate No 1470765, inventor V. V. Archipov, 15.09.90. Bulletin No 34. This reference is incorporated by reference. The cells separated by cold incubation with dog serum spread 2-3 times faster than cells separated with trypsin. Moreover, the trypsin treated cell culture had anomalous giant cells after four subcultivations and after five subcultivations, cell growth stopped and the culture degenerated. Cells separated by cold incubation with dog blood serum grew at a normal rate and we did not observe any anomalous cells in the culture through eight subcultivations (further passaging was not performed) . Plasminogen was prepared from the dog serum by a modification of the affinity chromatography method of D.G. Deutch and E.T. Mertz (Science, v.170, p.p.1095-1096 (1970)) which is hereby incorporated by reference. The purity of plasminogen preparations was estimated by SDS- electrophoresis as described by U.K. Laemmly (Nature, v.??7, p.p. 680-685 (1970)), which is hereby incorporated by reference. Plasminogen was identified by its N-end amino acid sequence by an automatic Edman degradation (about 30 residues) . The partial primary structure of dog plasminogen is reported by Y. Schaller, C. Straub, U.
Kampfer, E.E. Rickli, Complete Amino Acid Sequence Of Canine "Miniplasminoαen" , 2 Protein Seq. Data Anal. 445- 50 (1989) .
The sequence of human plasminogen is reported in L. Sottrup-Jensen, H. Claeys, M. Zajdel, T.E. Petersen, S. Magnusson in Progress in chemical fibrinolysis and thrombolysis (J.F. Davidson, R.M. Rowan, M.M. Samama, P.C. Desnoyers, eds) , v.3, p.p.191-209, Raven Press, New York (1978) and W. Wiman, Eur. J. Biochem. , v.76, p.p. 129-137 (1977).
The primary structure of bovine plasminogen is reported in J. Schaller, P.W. Moser, G.A.K. Dannegger- Muller, S.J. Rosselet, U. Kampfer and S.E. Rickli, Complete amino acid sequence of bovine plasminogen (comparison with human plasminogen) , Eur. J. Biochem., V.149,. p.p. 267-278 (1985). These references on the primary structure of plasminogens are hereby incorporated by reference.
We sterilized a saline buffer solution of plasminogen by filtering it through an 0.22 mm membrane filter. The buffer had a 1 mg/ml plasminogen protein concentration. To separate the cells from their substrate, we replaced the medium with serum free media and added plasminogen solution to the new media. The volume of plasminogen solution we added was between about 0.01 to about 0.1 volumes of the fresh medium. Our dog plasminogen working solutions had a plasminogen concentration, in the media, in the range of about 10 to about 50 μg/ml. We found that the amount of plasminogen necessary depends on the method of cell separation. For example, cold incubation (+4°C) requires one concentration, warm incubation (+37°C) another, and a warm/cold combination, such as described be"i<">w. yet another. 1. Incubating the culture for 4-5 hours at +37°C after adding the plasminogen solution required a minimum working dog plasminogen concentration of about 50μg/ml.
2. After adjusting the plasminogen concentration to at least about 10 μg/ml, we incubated the culture for 4-5 hours at +37°C. Thereafter, we cooled the culture to about 4°C and continued the incubation for about 12-24 hours.
3. Incubating the culture for about 24 hours at about +4°C after adding the plasminogen solution required a minimum dog plasminogen concentration of about 20 μg/ml. We compared Syria hamster BHK-21 fibroblasts separated by: a) dog plasminogen in above described conditions, or b) 0.25% commercial buffered Trypsin solution under standard conditions such as those described in
R.L.P. Adams, Cell Culture for Biochemists. (Biomedical Press 1980) , which is hereby incorporated by reference. The BHK-21 cells separated using plasminogen spread 1.5 - 2 times faster than cells separated with trypsin after one subcultivation. After 8 subcultivations with trypsin, the BHK-21 cells were morphologically modified (Fig. 2) . The trypsin treated cells spread less and did not form a continuous monolayer. This process is gradual. To 8th subcultivation these rejections are clear observed. After 10 subcultivations, the cells had a degenerative form and cell growth slowed down abruptly. After the 12th subcultivation, the culture degenerated (Fig. 3) .
We did not observe morphology modifications or a loss of cell spreading speed after 19 subcultivations when we used dog plasminogen for cell detachment (Fig. 5) .
From our results, w^ concluded that cells detached with plasminogen are damaged less than cells detached using trypsin. Furthermore, the cytoplasmatic membrane is preserved intact, which is confirmed by a considerably higher spreading speed of the cells and from the absence
of modifications in cell morphology in the course of 19 subcultivations. (The cells were not submitted to further subcultivation.)
We conducted analogous experiments with plasminogen from other mammalian species, for example, bovine and human plasminogen. We prepared these plasminogens using the same procedure we used to isolate the dog plasminogen. We checked the purity of the isolated plasminogens by SDS-electrophoresis as described by Laemmly (Fig. 5a) .
We conducted similarly successful experiments using bovine and human plasminogen to detach of cells from their substrate. In these experiments, we substituted bovine and human plasminogens for dog plasminogen using the conditions described herein. However, as described in detail below, we used more concentrated working solutions of the bovine and human plasminogens.
Incubating a BHK-21 cell culture in the synthetic medium Dulbecco's Minimum Eagle's Medium ("DMEM") containing about 0.19 mg/ml bovine plasminogen for about 4.5 to 5.0 hours at about 37°C detached about 95% of the cells from the substrate. We suspended these cells by pipetting particles of monolayer.
We also incubated a BHK-21 cell culture with about 0.075 mg/ml human plasminogen solution for 5 hours at 37°C, and then for 18 hours at 4°C. This process detached 100% of the cells from their substrate as a monolayer film. We also suspended these cells by pipetting. Based on our experience with dog, bovine and human plasminogen, we anticipate that other mammalian plasminogens will work, including for example, rabbit, muππs, porcine, monkey (such as gibbon) , artiodactyla, other carnivores, Lagomorpha, perissodactyla and rodent. Thus, the mammalian plasminogens at some conditions (a temperature, a period of incubation, a plasminogen concentration) detach cells from their substrates. The
detached cells are viable, spread rapidly, and grow well. The dog plasminogen was more active than either bovine or human plasminogen.
We believe plasminogen is converted into plasmin in the cell culture under some conditions. Plasmin hydrolyses adhesive proteins which are believed to connect cells to the substrate. Some enzymes convert plasminogen into plasmin, two of which may be found in a cell culture: namely, tissue-type and urokinase-type plasminogen activators. Cells can adsorb them from the serum during a cell grow. Many cultured cells have receptors for either or both enzymes. Plasminogen activators may bind to cellular receptors and localize plasmin formation. (See D.P. Beebe, Thromb. Res., v. 46, p.p. 241-254 (1987); K.A. Hajjar, N. M. Hamel, P. C.
Harpel and R. L. Nachman, J. Clin. Invest., v. 80, p.p. 1712-1719 (1988); F. Blassi, Fibrinolysis, v. 2, p.p. 73- 84 (1988); S. Stack, M. Gonzalez-Gronow, S. V. Pizzo, Biochem., v. 29, p.p. 4966-4970 (1990); E.S. Barnathan, A. Kuo, H. Van de Keyl, K. R. McCrae, G.R. Larsen, D. B. Cines, J. Biol. Chem. , v. 263. p.p. 7792-7799 (1988); E. S. Barnathan, A. Kuo, L. Rosenfeld, K. Kariko, M. Leski, F. Robbiat, M. L. Nolli, J. Henkin and D.B. Cines, J. Biol. Chem., v. 265, p.p. 2865-2872 (1990); and K. A. Hajjar and N. M. Hamel J. Biol. Chem., v. 265, p.p. 2908- 2916 (1990) .
Moreover, the literature suggests that some cells (for example, endothelial cells) synthesize and secrete both tissue-type and urokinase-type plasminogen activators. (See E. G. Levin D. J. Loskutoff, J. Cell. Biol., v. 94, p.p. 631-636 (1982); F.M. Boose, G. Osikowicz, S. Feder, J. Scheinbuks, J. Biol. Chem., v. 259, p.p. 7198-7205 (1984); F. Bachmann, E.K.n. ruithof, Semin. Thromb. Haemostasis, v. 10, p.p. 6-17 (1984); P.G. McGuire, N. W. Seeds, J. Cell Biochem., v. 40, p.p. 215-227 (1989); V.W.M. Va Hinsberg, Haemostasis, v. 18, p.207 (1988) ; D. A. Hart, A. Rehemtulla, Comp. Biochem.
Physiol., v. 90B, N4 p.p. 691-708 (1988) .
Initial mammalian plasminogens concentrations for detaching cells from their substrate are about the same as the concentrations of the plasminogen in the blood in the species from which the plasminogen is isolated.
Cells treated with plasminogen can be detached from the substrate both as a continuous film (Fig. 6) and as an individual cell suspension (Fig. 7) depending upon the incubation conditions and the medium's plasminogen concentration. Plasminogen (or plasmin) appears to act with high specificity on adhesive molecules without damaging cell membranes. Plasminogen detached cells can undergo prolonged passaging of cells, preserving their typical morphology (and presumably, their cell membranes) . Based on primary-isolated dog aorta endothelial cells and constant fibroblast Syria hamster BHK-21 line, we showed that plasminogen (or plasmin) detaches cells, and cells detached this way undergo significantly more cell passages (the upper limit was not tested) than cells detached using commercial trypsin solution.
EXAMPLE 1: Dog plasminogen
Each of the Example l protocols used BHK-21 cells that were cultured in 50 ml plastic flasks (Greiner company) under standard conditions (DMEM with 10% bovine serum) . A. WARM INCUBATION
Once the BHK-21 cells formed a monolayer (Fig. 1) , we removed the medium and added 6 ml of fresh, serum-free medium (DMEM from Sigma) . We added 0.3 ml of dog plasminogen solution (1 mg/ml) in a saline buffer to the monolayer in serum-free medium. We put these flasks in a C02 incubator at 37°C for 4.5 hours. After this incubation, we gently shook the flasks. The cell monolayer separated from the substrate (Fig. 6) . We pipetted the flask content to suspend the cells (Fig. 7) , added 1.7 ml of serum free DMEM to the flask and
transferred 1 ml of this cell suspension (a subcultivation coefficient of 1:8) to a new flask containing 7.2 ml DMEM and of 0.8 ml of bovine serum. We grew the cells in the new flask in a C02- incubator at 37°C until a monolayer was formed, at which time we repeated the process.
B. WARM/COLD INCUBATION
Once the BHK-21 cells formed a monolayer, we removed the medium and added 6 ml of fresh, serum-free medium (DMEM from Sigma) . We added 0.06 ml of dog plasminogen solution (1 mg/ml) in a saline buffer to the monolayer in a serum-free medium. We incubated this culture in a C02- incubator first at +37°C for 5 hours and then at +4°C for 15 hours. After this incubation, we gently shook the flask and separated the monolayer. We pipetted the flask content to suspend the cells, added 1.7 ml serum-free medium DMEM. We transferred l ml of cell suspension to a new flask - containing 7.2 ml DMEM and 0.8 ml of bovine serum. We grew the cells in the new flask in C02-incubator at 37°C until a new monolayer was formed, at which time we repeated the process.
C. COLD INCUBATION
Once the BHK-21 cells formed a monolayer, we removed the medium and added 6 ml of fresh, serum-free medium (DMEM from Sigma. We added 0.12 ml of dog plasminogen solution (1 mg/ml) in a saline buffer to the monolayer in serum-free medium. We incubated this culture in a C02- incubator at +4°C for 24 hours. After this incubation, we gently shook the flask and separated the monolayer. We pipetted the flask content to suspend the cells and added 1.7 ml serum-free medium DMEM. We transferred 1 ml of ce.l1 suspension to a new flask containing 7.2 ml DMEM and 0.8 ml of bovine serum. We grew the cells in a C02-incubator at 37°C until a new monolayer was formed. At that time, we repeated the process.
As bovine serum inhibits the activity of plasminogen, we did not need to remove the plasminogen during growth phases.
Based on light microscopy, we were able to perform 8 subcultivations of dog endothelial cells and 19 subcultivations of the BHK-21 cell line (both with plasminogen) without any visible change in the cellular morphology (Fig. 5) . EXAMPLE 2: Bovine Plasminogen Once the cells formed a monolayer, we removed the medium and added 6 ml of fresh, serum-free medium (DMEM from Sigma) . We added 0.19 ml of bovine plasminogen solution (1 mg/ml) in a saline buffer to the monolayer in serum-free medium. The working concentration of bovine plasminogen was 0.038 mg/ml. We incubated monolayer plus bovine plasminogen combination in a C02-incubator, first at 37°C for 5 hours and then at +4°C for 15 hours. After this incubation, we gently shook the flask and separated the monolayer. We pipetted the flask content to suspend the cells and added 1.7 ml serum-free medium DMEM. We transferred 1 ml of cell suspension to a new flask containing 7.2 ml DMEM and 0.8 ml of bovine serum.
We grew the cells in C02-incubator at 37°C until a monolayer was formed. At that time, we repeated the process.
EXAMPLE 3: Human Plasminogen
In this Example, after the formation of a BHK-21 cell culture monolayer, we changed the culture media to 6 ml of fresh serum-free DMEM media (from Sigma) .
Thereafter, we added 0.45 ml of human plasminogen solution (1 mg/ml) in a saline buffer (typically, a 0.01 M Na2HP04, pH 8.3 saline buffer) to the fresh, serum-free medium. Following the addition of plasminogen, the medium had a 0.075 mg/ml plasminogen concentration. We incubated the culture in a C02-incubator at 37°C for 5 hours, followed by a at 4°C incubation for 15 hours.
After the incubation, we gently shook the flask and separated the monolayer. We pipetted the flask contents to obtain a suspension of the individual cells and added another 1.7 ml of fresh, serum-free DMEM to the suspension. We then transferred 1 ml of this cell suspension into a new flask containing 7.2 ml of DMEM and 0.8 ml of bovine serum. We grew the cells in a C02- incubator at 37°C until a new monolayer was formed. At that time, we repeated the process. In each of the above examples, the plasminogen concentration reported is the minimum necessary to constituently detach 100% of the cells under the reported conditions. Lower plasminogen concentrations, shorter incubation times and lower incubation temperatures produced less than 100% cell detachment. Higher plasminogen concentrations, longer incubation times and higher incubation temperatures produced no discerned improvement in cell detachment. EXAMPLE 4: - (Prophetic Dog Prostheses Example) A sample of endothelial cells from a dog is surgically removed and aseptically transferred to an appropriate primary explanation cell culture medium. See, e.g., J. Paul, CELL & TISSUE CULTURE, which is hereby incorporated by reference. The cells are grown under appropriate aseptic conditions through several subcultivations. After several subcultivations, a dog blood-vessel prostheses is placed in the flask and used as a growth substrate. After a monolayer of endothelial cells has formed on the prostheses, the prostheses is surgically implanted in the dog from which the endothelial cells had been removed.
In an alternative embodiment, a monolayer of endothelial cells from a primary dog explanation cell culture is carefully detached from a growing flask and introduced between the threads of a blood-vessel prostheses. The cells are grown on the prostheses until they substantially cover the prostheses' surface. At
that time, the prostheses is surgically implanted in the dog from which the endothelial cells had been removed. EXAMPLE 5: - (Prophetic Human Prostheses)
The procedure of example 4 is repeated with a human patient. EXAMPLE 6: - (Prophetic Tissue Transplant)
The procedure of example 4 is repeated using a liver from another dog instead of a prostheses.
Claims
1. A method of separating a monolayer of cells from its substrate comprising the step of adding a mammalian plasminogen to said monolayer at a plasminogen concentration effective to separate said monolayer from said substrate in a medium substantially free of non-plasminogen proteolytic enzyme activity.
2. A method according to claim 1 employing a plasminogen selected from the group consisting of dog plasminogen, bovine plasminogen, human plasminogen and combinations thereof.
3. A method according to claim 1 which further comprises incubating said monolayer in the presence of said plasminogen at a temperature of at least about 25°C for at least about two hours.
4. A method according to claim 3 which further comprises incubating said monolayer in the presence of said plasminogen at a temperature of less than 15°C for at least about six hours.
5. A method according to claim 1 which further comprises incubating said monolayer in the presence of said plasminogen at a temperature of less than 15°C for at least about six hours.
6. A method of separating a monolayer of cells from its substrate comprising increasing the plasmin activity in the medium surrounding said cells to a level effective to separate said monolayer from said substrate.
7. A method according to claim 6 wherein said plasmin activity in said medium is increased by a step comprising increasing the activity of a plasminogen activator present in said cells and medium.
8. A method according to claim 7 in which said medium is substantially serum-free.
9. A method according to claim 7 wherein the activity of said plasminogen activator is increased by a step comprising adding a co-factor.
10. A method according to claim 7 wherein the activity of said plasminogen activator is increased by a step comprising adding a modulator.
11. A method according to claim 7 wherein the activity of said plasminogen activator is increased by a step comprising adding a plasmin precursor.
12. A method according to claim 11 wherein said plasmin precursor comprises a mammalian plasminogen.
13. A method according to claim 12 wherein said mammalian plasminogen comprises dog plasminogen.
14. A method according to claim 13 which further comprises incubating said monolayer of cells in the presence of said dog plasminogen at a temperature of between about 25°C and about 45°C for at least about 30 minutes.
15. A.method according to claim 14 which further comprises a second incubation of said monolayer of cells in the presence of said dog plasminogen at a temperature of between about
4°C and about 15°C for at least about six hours.
16. A method according to claim 13 which further comprises incubating said monolayer of cells in the presence of said dog plasminogen at a temperature of between about 4°C and about 15°C for at least about six hours.
17. A method of separating a monolayer of cells from its substrate comprising incubating said monolayer in the presence of dog plasminogen at a plasminogen concentration, for a time and at a temperature sufficient to separate said monolayer from said substrate.
18. A method according to claim 17 which is substantially free of proteolytic enzymes other than plasmin.
19. A composition for separating a monolayer of cells from its substrate comprising a member of the group consisting of mammalian plasminogens, mammalian plasmins and combinations thereof and a carrier, said member of the group being present in said carrier at a concentration capable of separating said monolayer from said substrate when added to a cell medium.
20. A method of reducing the risk of rejection by a recipient of a surgical implant, while minimizing the introduction of transformed cells into a recipient, comprising: a) preparing a primary cell culture of cells from the recipient of said implant; b) culturing said primary cell culture until it forms a substantial biomass; c) detaching the biomass from its substrate; and d) inserting said implant into said recipient.
21. A method according to claim 20 further comprising detaching said cells from said recipient from their attachments using plasminogen.
22. A method according to claim 20 further comprising detaching a subcultavation of said primary cell culture from its substrate using plasminogen.
23. A method according to claim 20 wherein said recipient is a dog.
24. A method according to claim 20 wherein said recipient is a horse.
25. A method according to claim 20 wherein said recipient is a human.
26. A method of reducing the risk of rejection by a recipient of a surgically implanted protheses, while minimizing the introduction of transformed cells in^c a recipient, comprising: a) preparing a primary cell culture of cells from the recipient of said implant; b) culturing said primary cell culture until it forms a substantial biomass; c) binding an average of at least about 102 cells from said primary cell culture on each mm2 of said protheses; d) culturing said primary cell culture on said protheses until there are at least about 102 to 10* cells per mm2 of surface area of said protheses; and e) inserting said protheses into said recipient.
27. A method according to claim 26 further comprising detaching said cells from said recipient from their attachments using plasminogen.
28. A method according to claim 26 further comprising detaching a subcultavation of said primary cell culture from its substrate using plasminogen.
29. A method according to claim 26 wherein said recipient is a dog.
30. A method according to claim 26 wherein said recipient is a horse.
31. A method according to claim 26 wherein said recipient is a human.
32. A prostheses having a reduced rate of rejection comprising: a. a protheses; and b. a layer of cells derived from recipient of said protheses bound to the surface of said protheses.
33. A protheses according to claim 32 in which said cells are anchorage dependent cells.
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SU925065876A RU2039816C1 (en) | 1992-08-05 | 1992-08-05 | Preparation for detaching contact-dependent cells from substrate |
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US5741685A (en) * | 1995-06-07 | 1998-04-21 | Children's Medical Center Corporation | Parenchymal cells packaged in immunoprotective tissue for implantation |
SE9703144L (en) * | 1997-09-01 | 1999-03-02 | Cardia Innovation Ab | Preparation, method of making a preparation, use of a preparation for treatment and method of treatment |
AU9253898A (en) * | 1997-09-26 | 1999-04-23 | Novo Nordisk A/S | Use of proteases in passaging of adherent animal or human cell cultures |
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US4177262A (en) * | 1974-12-17 | 1979-12-04 | Choay S.A. | Plasminogen compositions containing preactivated plasminogens with or without native plasminogens, process for making same, pharmaceutical compositions and control of blood clots |
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