Patent Application
20090042289
The present invention relates to methods for preserving cells, in particular primary cells, such as hepatocytes. The present invention also relates to a gel containing hydrolysed gelatin.
The introduction of whole organ collagenase perfusion techniques (Krebs et al., Regulation of Hepatic Metabolism, 726-75, 1974: and Seglen, Meth. Cell Biol., 13, 29-83, 1976) has lead to the widespread isolation of viable hepatocytes. The isolation methods used are mild with the result that the cells initially retain in vivo like properties in vitro. However, high cell yields are obtained from a single liver and generally the number of cells produced greatly exceeds the experimental requirements.
Isolated hepatocytes are a better approximation to the in vivo situation than established cell lines or hepatoma cells (Sirica and Pitot, Pharmacol. Rev., 31, 205-227, 1980) but they have the disadvantage of a limited viability in suspension culture.
Hepatocytes have many features which make them a particularly suitable in vitro model system. They have a high rate of metabolism and are the major cell type involved in detoxification. However, in culture the cells divide poorly, if at all, and undergo phenotypic changes whereby many of their liver specific processes are lost. The cells are generally used immediately after isolation and sustained viability is dependent upon the formation of a monolayer. Maintenance of large numbers of plates is labour intensive as the medium needs to be changed every 24 hours.
A further complication during hepatocyte culture is their limited life span and the occurrence of phenotypic changes (Bissell, Ann. N.Y. Acad. Sci., 349, 85-98, 1980). These changes make results obtained using long-term cultures no more applicable to the intact organ than those obtained with established cell lines.
The hypothermic preservation of hepatocytes on gelatin gels has been disclosed (Evans, Cell Biology International, 18, 999-1008, 1994; Evans, Cell Biology International, 19 855-860, 1995; Evans, Cell Biology International, 23, 117-124, 1999; and Griffiths & Evans, Cryobiology, 40, 176-181, 2000). The method requires the preparation of gels made from a culture medium containing untreated (non-hydrolysed) gelatin and the attachment of hepatocytes to the gel surface. The hepatocytes are then overlaid with culture medium and cultured at a temperature of 10° C. Such preservation of hepatocytes on gelatin containing gels enables hepatocytes to be stored for at least 7 days without any substantive deterioration in the function of the cells. In order to detach the cells from the gel surface, it is necessary to melt the gel by heating it to 37° C. Heating the gel to 37° C. may cause damage to the cells. A further problem with this prior art method is that the culture medium overlying the attached cells needs to be changed. As the cells are attached to an exposed surface of the gel, the cells may be dislodged or damaged when the medium is changed. Furthermore, on transporting a culture dish, the culture medium can easily be spilled.
U.S. Pat. No. 5,635,344 and U.S. Pat. No. 5,736,397 disclose a method of preserving cells comprising culturing the cells at a temperature of between 0 and 5° C., wherein the cells are in a gel comprising non-hydrolysed gelatin. Culturing cells between layers of non-hydrolysed gelatin in not disclosed. The present invention overcomes at least some of the problems associated with the prior art methods for preserving cells.
The present invention provides a method for preserving isolated cells comprising attaching the cells to a gel comprising hydrolysed gelatin and culturing the cells at a temperature of between 0° C. and 5° C.
The use of hydrolysed gelatin produces a gel which is strong enough to act as a support to which cells can be attached, but weak enough to be dispersed by the addition of an aqueous solution such as excess cold culture medium. Rimming (i.e. use of a needle or similar device to facilitate separation of gel from its container) can also be used to assist with the dispersion.
The use of hydrolysed gelatin in the gel therefore avoids the need to heat the gel to 37° C. in order to detach the cells and avoids any damage to the cells caused by such heating. A distinction can also be made between hypothermic damage and damage caused by heating to 37° C. by using the gel comprising hydrolysed gelatin.
Furthermore, by using hydrolysed gelatin in a gel, the attachment of cells to a solid support (i.e. a standard tissue culture treated plastic support such as a culture dish or well, e.g. Primaria® and Corning) is inhibited provided the gel does not contain serum. The solid supports have been treated so that they have a net charge on their surface in order to aid in the attachment of cells to the support. It has been found that the hydrolysis of gelatin results in the loss of factors which encourage cells to attach to a solid support and/or generates factors that inhibit cell attachment. Accordingly, the method of the present invention enables one skilled in the art to prevent the attachment of cells to a solid support and thereby enables easier harvesting of the cultured cells and in particular facilitates the isolation of spheroids. Spheroids are cells that form a multicellular aggregate. The aggregate is usually spherical. The spheroids can reconstruct functional complexes and show tissue specific functions (see Hamamoto et al., J. Biochem., 124, 972-979, 1998).
As the presence of hydrolysed gelatin inhibits the attachment of cells to solid supports when serum is not present, it allows one skilled in the art more time to recover cells without the complication of cell attachment to a solid support. The absence of serum assists in preventing cells attaching to a solid support. The absence of serum from the medium leads to a decrease of the period in which the cells can be preserved from about 7 days to 3 days. It is also preferred that the solid support does not have a coating of an extracellular matrix factor, such as collagen, fibronectin and matrigel. Such extracellular matrix factors help cells to attach to the solid support and should be avoided if the cells are not to be attached to the surface of the solid support.
Furthermore, by using hydrolysed gelatin in a gel, it has been found that the gel melts uniformly at about 37° C. On heating a gel containing hydrolysed gelatin, the gel melts more uniformly than a gel containing non-hydrolysed gelatin. The term “uniformly” as used herein means the centre of the gel melts at substantially the same rate as the edges of the gel. This uniform melting of the gel has the advantage that the all the cells can be isolated from the gel at substantially the same time.
The term “preserving” as used herein is defined as maintaining the viability and/or in vivo-like function of the cells. Viability can be judged by the retention of cytosolic marker enzymes such as lactate dehydrogenase (LDH) and by the maintenance of the cells' ability to subsequently attach to a solid support under non-inhibitory culture conditions. Retension of in vivo-like function can be judged by the cells maintaining rates of protein synthesis, by cells demonstrating in vivo-like responses to hormones, by cells maintaining the level of cytochrome P450 and its specific isoenzymes, and by the absence of atypical enzyme characteristics or phenotypic changes. More than one of these methods can be used to determine in vivo-like cell function.
Preferably in vivo-like cell function is measured by determining that the levels of cytochrome P450 are maintained. Methods for determining the viability and in vivo-like cell function of cells are disclosed in Evans, 1994 (supra), Evans, 1995 (supra), Griffiths and Evans, 2000 (supra) and Evans, 1999 (supra).
The term “attaching” as used herein refers to the cells being immobilised on a surface of the gel or immobilised within the gel.
The term “isolated cell” as used herein, refers to any cell except gelatinase producing bacteria. Preferably, the isolated cell is a eukaryotic cell such as a mammalian cell, a plant cell (including protoplasts), insect cells, etc. It is particularly preferred that the term refers to primary cells such as hepatocytes, kidney proximal tubule cells, mammary epithelial cells and primary pancreatic cells. The isolated cell may also be a genetically engineered cell containing one or more heterologous genes or modified genes. Preferably the isolated cell is a hepatocyte obtained following whole organ collagenase perfusion or an equivalent method. Methods for obtaining isolated hepatocytes are well known to those skilled in the art. In particular, suitable methods are disclosed in Evans (Biochim. Biophys. Acta, 677, 433-444, 1981) and Evans and Mayer (Biochim. Biophys. Res. Comm., 107 51-58, 1982).
Methods for obtaining kidney proximal tubule cells are described in Evans (Biochem. Biophys. Acta, 1133, 255-260, 1992) and Evans (Biochem. Biophys. Acta, 1221, 243-249, 1994). It has been shown that on preserving kidney proximal tubule cells using the method of the present invention that in addition to maintaining the in vivo like enzyme profiles, the kidney proximal tubule cells divide normally when released from the gel. The conditions of preservation have no cystostatic effects.
The method of the present invention is therefore appropriate for both non-dividing and dividing cell types. Moreover, the kidney proximal tubule cells rely almost exclusively on aerobic respiration for their energy generation while the hepatocytes have a significant capacity for anaerobic respiration. The successful preservation of these primary cell type, which have very different characteristics show that the method of the present invention is applicable to a wide variety of cell types. The method of the present invention is a very valuable holding procedure for primary cells and is also appropriate for use with established cell lines. In particular, it obviates the need to “grow cells up” immediately prior to use. Experiments requiring confluent cell monolayers, e.g. Caco 2 cells, can therefore also be facilitated.
The term “culturing” as used herein refers to the maintenance and/or growth of cells.
Any gel containing hydrolysed gelatin can be used in the method of the present invention provided it is suitable for culturing the cells. The gel preferably comprises a cell culture medium and hydrolysed gelatin. The cell culture medium can be any cell culture medium capable of culturing the cells to be preserved. A suitable culture medium is Leibovitz (L-15) medium preferably buffered with Hepes in place of bicarbonate enabling it to be exposed to the atmosphere. Other suitable media include Waymouth's, Williams', Dulbecco's or Ham's medium which are buffered with bicarbonate and require a 95% air 5% CO2 gas phase. Where necessary, the media can be supplemented. Suitable supplements include carbon sources such as glucose, growth hormones such as insulin, serum such as heat inactivated new born calf serum and antibiotics such as gentamicin. Preferably the gel comprises 1.5% (w/v) hydrolysed gelatin in Leibovitz (L-15) medium (pH 7.4) containing glucose (8.3 mM) and Hepes (25 mM).
The cell culture medium may in addition comprise a cytoprotectant, such as glutathione, to protect the cells against rewarming injury. Glutathione is known to enter cells at low temperatures.
Preferably, the method of the present invention comprises culturing the cells at a temperature of between 4 and 14° C., more preferably at a temperature of between 8 and 12° C., most preferably at a temperature of about 10° C.
The term “hydrolysed gelatin” as used herein means gelatin that has been at least partially hydrolysed to reduce its gel strength. Gelatin that has not been hydrolysed produces a relatively strong gel. When using hydrolysed gelatin, a weaker gel is formed that can be dispersed by the addition of an aqueous solution. Preferably the term “hydrolysed gelatin” as used herein, refers to gelatin which is hydrolysed to a degree such that it will produce a gel which is sufficiently strong to provide a solid support to which cells can be attached at a temperature of between 0 and 15° C., but which is sufficiently weak to be dispersed on addition of an aqueous solution. Preferably the hydrolysed gelatin is hydrolysed 300 bloom type A gelatin from porcine skin.
Gelatin can be hydrolysed by any known method including thermal hydrolysis, proteolytic digestion with gelatinase or trypsin, and chemical methods including acid and alkaline hydrolysis. Preferably the gelatin is hydrolysed by thermal hydrolysis. In particular, it is preferred that hydrolysed gelatin is produced by heating gelatin to a temperature of between 85 and 121° C. for between 0.2 and 3 hours. More preferably, the gelatin is heated to a temperature between 90 and 100° C. for 1 to 2.5 hours. Most preferably, the gelatin is hydrolysed by heating it to about 95° C. for about 2 hours or by heating it to about 121° C. for about 20 minutes.
The gel preferably comprises sufficient hydrolysed gelatin to produce a gel that is sufficiently strong to produce a solid gel on, or in, which cells can be preserved at a temperature of between 0 and 15° C., but which is sufficiently weak to be dispersed on addition of an aqueous solution. Preferably, the gel comprises between 0.5% and 5.0% (w/v) of hydrolysed gelatin. It is further preferred that the gel comprises between 1% and 2% (w/v) hydrolysed gelatin, most preferably 1.5% (w/v) gelatin.
The gel may in addition to the hydrolysed gelatin comprise non-hydrolysed gelatin, provided the gel formed is sufficiently weak to be dispersed by addition of an aqueous solution at a temperature of between 0 and 15° C.
In a preferred embodiment of the present invention, the cells are attached to a surface of the gel comprising hydrolysed gelatin and preferably covered with a liquid cell culture medium. Generally the liquid cell culture medium is identical to that used to form the gel except that it does not comprise gelatin or hydrolysed gelatin and may comprise supplements not present in the gel. It is particularly preferred that the liquid cell culture medium comprises a cytoprotectant, such as glutathione. In a further embodiment of the present invention, the cells are attached to a first layer of the gel comprising hydrolysed gelatin and then covered with a second layer of the hydrolysed gelatin gel so that the cells are held between the two layers of gel. In an alternative embodiment of the present invention the cells are mixed with the gel comprising hydrolysed gelatin before it solidifies (i.e. at a temperature of about 37° C.). When the gel solidifies (i.e. at a temperature of about 10° C.) the cells are immobilised with the gel. If necessary, a liquid cell culture medium can be added in order to prevent the gel drying out and to supply any required nutrients. By having the cells between two layers of the gel or immobilised within the gel, the cells are protected from damage during transport and it is also easier to change the liquid culture medium without disturbing the cells. On dispersing the gel, the cells may attach to the surface of the solid support, in which the gel is formed, provided the gel contains serum and/or the surface of the solid support is coated with an extracellular matrix factor. Alternatively, the cells may be prevented from attaching to the solid support by using a gel which does not contain serum and preferably by using a solid support that has not been coated with an extracellular matrix factor.
In a further preferred embodiment of the present invention, the cells can be attached directly to a solid support, such as culture dish or well and then covered with a layer of the gel comprising hydrolysed gelatin. The cells will then be covered by the gel. If necessary, the gel can then be covered with cell culture medium in order to prevent the gel drying out and to supply any required nutrients. Again this protects the cells from damage during transport and on changing the cell culture medium. Furthermore, if it is desired to use the cells when they are attached to a surface, this method will enable the cells to be used directly after the gel has been dispersed without waiting for cell attachment. Preferably the surface of the solid support has a coating of an extracellular matrix factor. Furthermore, the hydrolysed gelatin gel layer can be covered with a non-hydrolysed gel layer. Preferably the non-hydrolysed gel layer is thin, i.e. approximately between 0.5 and 2 mm thick. The top non-hydrolysed gelatin gel layer can act as a more robust top layer for protection during distribution. The term “non-hydrolysed gelatin” is defined below.
The method of the present invention can be performed using any suitable solid support for supporting the gel. Suitable solid supports include culture dishes, wells such as 96 well format plates and columns. For example, the method can be used to produce solid supports having cells affixed to their surface ready for use in standard screening or biochemical assay procedures well known to those skilled in the art as indicated above.
It is surprising that it is possible to cover the cells with the gel as it was known that cells can easily be damaged by shear forces. Shear forces are used to break open cells in a controlled manner by homogenation, sonification and nitrogen cavitation. The gelation of gelatin would have been expected to exert strong forces on cells in its vicinity and possibly to remove water from the cells. It was therefore surprising to find that on covering and immobilising cells within the gel that no cytotoxic effects occurred.
The present invention also provides a gel comprising hydrolysed gelatin for use in the method of the present invention. As indicated above, any culture medium can be used in combination with hydrolysed gelatin to form the gel of the present invention.
As indicated above, the gel of the present invention has the advantage of enabling the preservation of cells at temperatures of between 0 and 15° C. wherein the cells can be detached from the gel by dispersing the gel on addition of an aqueous solution.
The present invention also provides a solid support having the gel of the present invention formed thereon.
The solid support can be any support on which the gel can be formed. In particular, the solid support can be a culture dish, a well or a column. The solid support may have a coating of an extracellular matrix factor.
It is further preferred that cells are attached to the gel formed on the solid support of the present invention. As indicated above, the cells can be:
The present invention also provides a gel comprising hydrolysed gelatin and one or more isolated cells.
The present invention also provides a method for preserving isolated cells comprising attaching the cells to a first layer of a gel and then covering with a second layer of a gel so that the cells are held between the two layers of gel and culturing the cells at a temperature of between 0 and 15° C., wherein at least one of the layers of gel comprises non-hydrolysed gelatin.
The term “non-hydrolysed gelatin” refers to gelatin which has not been hydrolysed. Preferably the term “non-hydrolysed gelatin” means gelatin which has not been hydrolysed in vitro. It is further preferred that the gelatin has not been hydrolysed by thermal hydrolysis, proteolytic digestion with gelatinase or trypsin or chemical methods including acid or alkaline hydrolysis.
Preferably the gelatin is 300 bloom type A gelatin from porcine skin which has not been hydrolysed.
In a preferred embodiment, both layers of gel comprise non-hydrolysed gelatin.
In an alternative preferred embodiment, the first layer of gel comprises hydrolysed gelatin and the second layer comprises non-hydrolysed gelatin.
In a further preferred alternative embodiment, the first layer of gel comprises non-hydrolysed gelatin and the second layer comprises hydrolysed gelatin. When the cells are required, the second layer can be removed by aqueous dispersion, leaving the cells still attached to the first layer. Such immobilised cells are particularly suited for microinjection studies. Various substances (e.g. DNA, RNA and proteins) can be directly injected into the immobilised cells. In view of the low metabolism rate of the cells, the progressive microinjection of the immobilised cells will give essentially a uniform starting time for subsequent metabolic experiments.
The gel comprising the non-hydrolysed gelatin may in addition also comprise hydrolysed gelatin; however, preferably the gel comprising non-hydrolysed gelatin contains substantially no hydrolysed gelatin. Substantially no hydrolysed gelatin mean the gel does not contain more than the endogenous level of hydrolysed gelatin present in a non-hydrolysed gelatin sample. Preferably the level of hydrolysed gelatin is less than 10% (w/w) of the gelatin. Hydrolysed gelatin is considered to be gelatin protein that is soluble in trichloroacetic acid. Accordingly, it is preferred that non-hydrolysed gelatin contains less than 10% (w/w) tricloroacetic acid soluble protein. Preferably the gel comprising non-hydrolysed gelatin forms a strong gel on which cells can be preserved at a temperature of between 0 and 15° C., but which cannot be dispersed on addition of an aqueous solution. Suitable non-hydrolysed gelatin gels have been described previously (see Evans, Cell Biology International, 18, 999-1008, 1994; Evans, Cell Biology International, 19, 855-860, 1995; Evans, Cell Biology International, 23, 117-124, 1999; and Griffiths & Evans, Cryobiology, 40, 176-181, 2000). Furthermore, hydrolysed gelatin is generally found to comprise a 30 kDa protein fragment whereas non-hydrolysed gelatin does not comprise a 30 kDa protein fragment (as detected by using SDS-PAGE).
The preferred features of the gelcomprising non-hydrolysed gelatin including the various components of the gel are the same as those indicated above for the gel comprising hydrolysed gelatin, except that the gel must comprise non-hydrolysed gelatin. The present invention also provides a solid support having a first layer of gelatin gel formed thereon and a second layer of gelatin formed on the first layer of gelatin gel, wherein cells are held between the two gelatin gel layers. The gelatin gel used to form the first and second layer may be independently selected from a hydrolysed gelatin gel or a non-hydrolysed gelatin gel.
The solid support and cells are as defined above.
This two layer configuration has never previously been successfully used to preserve cells hypothermically. Indeed the only case where it has been tried (Stevanovich et al., Cryobiology, 32, 389-403, 1995), it leads to rapid membrane blebbing and cell death.
The two layer configuration of the present invention has a number of advantages. The first is that the two layer configuration enables a uniform cell monolayer to be formed on the lower gelatin layer prior to adding the upper gelatin layer. In contrast, storage in a cell suspension will not be uniform and may cause local high cell densities at the bottom of the container or possibly in the body of the solidifying gelatin. The rapid rate of cell sedimentation suggests that the effect will be most marked near and at the bottom of the container. Cell stacking is likely to also be a problem as the cells sediment. A further advantage relates to diffusion through gelatin gels. It must be remembered that metabolism is continuing under hypothermic conditions i.e. the cells are not in suspended animation. Energy is still required for maintenance purposes (e.g. ion pumps and protein degradation). The two layer configuration allows diffusion across the entire cell surface unlike when the cells are either on the top of the surface of the gelatin gel or are attached to a solid support and then covered with the gelatin gel.
Furthermore, the depth of the gelatin gels above the cells is generally less than that when the cells are immobilised on a solid support and covered with the gel, again allowing better diffusion of nutrients to the cells.
Furthermore, by preserving cells between two layers, the cells will have uniform forces over their entire surface area. The cells will therefore distort less and are less likely to undergo phenotypic changes. Furthermore, the inventors have found that cells survive longer in a non-hydrolysed gelatin gel two layer configuration than when cells attached to a solid support are covered with a non-hydrolysed gelatin gel layer or when cells are immobilised within a non-hydrolysed gelatin gel.
Accordingly, there are numerous advantages associated with the two layer configuration for immobilising cells.
As indicated previously, it is desirable, although not necessary, to have the gelatin layers covered with a liquid culture medium. This is particularly preferred when the duration of preservation is over a long period of time.
The inventors of the present invention have also found that following long-term storage of cells at temperatures of between 0 and 15° C., organelles such a mitochondria aggregate to the centre of the cells (Griffiths et al., Cryobiology, 40, 176-181, 2000). This is due to the collapse of the cytoskeleton in the cell. It has been found that subjecting the cells to brief periods at temperatures or between 20 and 37° C., is sufficient to restore cytoskeleton structures while being too short to initiate phenotypic changes in the cells.
Preferably, the methods of the present invention additionally comprise subjecting the cells to temperatures of between about 20 and 37° C. for about 1 to 16 hours (preferably about 5 hours) at least once every few days (i.e. about every 2 to 5 days). However, if the increased temperature results in the gelatin gel melting (e.g. when a hydrolysed gelatin gel is subjected to a temperature of about 25° C. or when a non-hydrolysed gelatin gel is subjected to a temperature of about 37° C.) it is preferred, that the cells are transferred to a suspension culture for the duration of the increased temperature and then attached to a gelatin containing gel for continued preservation. Suspension cultures are well known to those skilled in the art. In particular, a suspension culture for hepatocytes is described in Evans, Biochim. Biophys. Acta., 677, 433-444, 1981.
The present invention also provides a method of preserving isolated cells comprising attaching the cells to a gel comprising non-hydrolysed gelatin and/or hydrolysed gelatin and culturing the cells at a temperature of between 0 and 15° C., and further comprising subjecting the cells to temperatures of between about 20 and 37° C. for about 1 to 16 hours (preferably about 5 hours) at least once every few days (i.e. about every 2 to 5 days). The cells may be attached to a surface of the gel, held between two layers of the gel, immobilised within the gel or attached to a solid support and covered with the gel.
The methods of the present invention enable “cell banks” of primary cells to be made from one animal. The cells can subsequently be used over a period of days, avoiding the complications of genetic variation. The methods enable cells to be used immediately without waiting for the cells to multiply. The methods enable the preservation of cell types which are sensitive to cryopreservation and allows genetically modified cells to be transported to distant locations with minimal losses.
The present invention also provides a method for preserving isolated organs or fragments of tissue comprising perfusing the organ or tissue with a liquefied gel comprising hydrolysed and/or non-hydrolysed gelatin, wherein subsequent to perfusion, the organ or tissue is stored at a temperature of between 0 and 15° C.
A major difficulty with organ transplantation is the short time period available before the organ deteriorates (e.g. 36-48 h for a liver). Many argue that this is not initially due to the poor preservation of the organ specific cells but the greater sensitivity of the endothelial cells lining the blood vessels (T. Ebert et al., Modern Trends in BioThermoKinetics, 3, 288-293, 1994). These cells “slough off” the walls, when reperfusion is attempted, leading to occlusion of the blood vessels and subsequent organ damage due to impaired circulation. Perfusion of a freshly isolated organ with a gelatin preservation medium prevents these changes. Following complete perfusion, the organ is stored at between 0 and 15° C. with the result that the gelatin solidifies within the blood vessels etc. and the endothelial cells are held in their correct positions. The gelatin gel combines the features of a solid supporting matrix, a good diffusion rate with the tissues and a low melting temperature. When the organ is required is can be perfused with a medium (e.g. saline) at 37° C. The preserving gelatin gel is washed out rapidly. The use of the gelatin has a further benefit that gelatin is a normal body material and is unlikely to be immunogenic.
The organ can be any organ such as a heart, liver, kidney etc. The tissue can be any tissue having vasulature capable of being perfused. Preferably, an organ is perfused and then tissue samples taken from the perfused organ. Alternatively, a tissue sample can be perfused directly provided it has sufficient vasculature to allow perfusion.
The liquefied gel is a gelatin gel which is liquefied by maintaining it at a sufficiently high temperature (e.g. 25 to 37° C.) to ensure that it does not solidify. Preferably the gelatin gel comprises hydrolysed gelatin or non-hydrolysed gelatin and is as defined above. Most preferably, the gelatin gel comprises hydrolysed gelatin as defined above.
The present invention will now be described by way of examples with reference to the following:
a shows the effect of culture (37° C.) on hepatocyte levels of cytochrome P450 and cytochrome b5 and
a shows the intracellular protein degradation in hepatocytes cultured at 37° C. and
Gel a: (i) MW: 95 KDa
Gel b: (i) MW: 95 KDa; (ii) MW: 30 KDa
Gel c: (i) MW: 30 KDa
Gel d: (i) MW: 30 KDa
a shows the temperature profile within different wells of a 96-well microplate during the cooling stage (10° C.). See
Collegenase was purchased from Boehringer Mannheim, Opti Phase X from LKB, Croydon, Surrey, U. K. Leibovitz (L-15) culture medium, new born calf serum and gentamycin were supplied by ICN by Biomedicals LTD, High Wycombe, Bucks U. K. and insulin was obtained from Boots, Nottingham, U K. All other chemicals were obtained from Sigma, Poole, Dorset, U K.
Hepatocytes were prepared as described previously (Evans 1981 (supra), Evans and Mayer, 1982 (supra)). The gel of the present invention comprises cell culture medium consisting of Leibovitz (L-15) medium (pH7.4) containing glucose (8.3 mM) and Hepes (25 mM), and hydrolysed gelatin. Hydrolysed gelatin was obtained by heating gelatin (3%, Sigma type I from porcine skin) in sterile water to 95° C. for 2 hours. One volume of the 3% hydrolysed gelatin solution was added to 1 volume of a 2× solution of the cell culture medium to form a solution containing 1.5% (w/v) hydrolysed gelatin. The gel containing the hydrolysed gelatin was allowed to cool to 10° C. in a 60 mm culture dish. 2.5×106 hepatocytes were added in 3 mls of Leibovitz (L-15) medium (pH7.4) supplemented with glucose (8.3 mM), Hepes (25 mM), insulin (0.8 μg ml−1), gentamicin (50 μg/ml−1) and heat inactivated new born calf serum (10% v/v). One hour was allowed for attachment of the cells to the gel at 10° C. The cells attached firmly to the gel but were not able to spread or migrate across the substratum. The plates were placed in a humidified 10° C. incubator.
The plates were sampled at daily intervals and found to maintain high viability as judged by their retention of the cytosolic marker enzyme lactate dehydrogenate (LDH) and their subsequent ability to attach to a solid support. The cells were also found to maintain their rates of protein synthesis and to show in vivo like responses to hormones. The levels of cytochrome P450 and its specific isoenzymes were found to be maintained. Furthermore, atypical enzyme characteristics of phenotypic changes did not appear.
The preserved hepatocytes maintained the above mentioned properties for at least seven days.
The hepatocytes were detached from the gel by addition of excess cold culture medium with rimming.
The hepatocytes detached from the gel were harvested and could be used in assays and other experiments.
The experiment described in Example 1 was repeated except that after attachment of the hepatocytes to the gel layer, the culture medium was removed and replaced with 1 ml of the L-15 cell culture medium comprising hydrolysed gelatin 1.5% (w/v). The upper gel layer was allowed to attach to the lower layer of gel for 1 hour at room temperature. The gel layers and cells where then placed at 10° C. to allow the gel to solidify. A further 1 ml of cell culture medium was added to the top of the upper gel layer. See
The preserved hepatocytes were found to be preserved in an identical manner to the hepatocytes of Example 1.
Again the cells were detached from the gel by addition of excess cold culture medium (with rimming).
The L-15 medium comprising 1.5% hydrolysed gelatin as described in example 1 above is melted at 37° C. and hepatocytes are added. The gel containing the hepatocytes is then placed at 10° C. so that the gel solidifies and the hepatocytes are immobilised within the gel (see
The preserved hepatocytes were found to be preserved in an identical manner to the hepatocytes of Example 1.
Again the cells were detached from the gel by addition of excess cold culture medium with rimming.
Hepatocytes are cultured in a petri dish in the L-15 medium described in example 1, which does not comprise 1.5% hydrolysed gelatin, at 37° C. for 2 hours. The hepatocytes attach to the surface of the dish. The L-15 medium is removed and replaced by the L-15 medium comprising 1.5% hydrolysed gelatin described in example 1. The dish is placed at 10° C. and the gel solidifies covering the cells attached to the surface of the dish. See
The preserved hepatocytes were found to be preserved in an identical manner to the hepatocytes of Example 1.
The gel was removed by heating or by the addition of excess cold culture medium with rimming and the cells remained attached to the surface of the dish ready for use in assays or other experiments.
As will be apparent to one skilled in the art, the amount of gelatin, solid support to which the cells can attach and the type of culture medium used to form the gel can vary depending on the exact requirements of the study. Examples 2 to 4 have also been performed using non-hydrolysed gelatin in place of hydrolysed gelatin and the cells detached from the gel by heating the gel to 37° C.
The functional integrity of preserved cells was examined using the following criteria:—
1. Over a 7 day period 90-95% of the cells released from the gelatin gels (both non-hydrolysed gelatin and hydrolysed gelatin gels) excluded the dye trypan blue. This figure is essentially the same as the viability of the freshly isolated cells.
2. Exclusion of vital dyes or retention of soluble enzymes indicates that the cells are alive at that instant. However, sustained viability is not necessarily indicated by these techniques. Poullain et al. (Hepatology 15, 97-106, 1992) reported that the lactate dehydrogenase activity after cold storage in the University of Wisconsin solution is relatively high even though the cells were unable to attach to plastic and survive. Similarly cryopreserved cells also often show these “contradictory” properties. Due to this uncertainty, cells preserved using the gelatin gels of the present invention were screened for their ability to attach to supports over a period of 2 hours at 37° C. and to express biochemical markers lactate dehydrogenase (LDH). The preserved cells were found to attach to supports in an identical manner to freshly isolated (non-preserved) cells. See
3. Attachment to a support is not conclusive proof that the hepatocytes will subsequently maintain their function at 37° C. Moreover, the cells may require a recovery period and have deteriorated metabolic function, as has been observed for cryopreserved cells (De Loecker et al. Cryobiology 30, 12-18, 1993; Chesne et al. Hepatology 18, 406-414, 1993). Accordingly, the rates of protein synthesis were used as a criterion of normal behaviour. Protein synthesis requires a high degree of metabolic integration. Typical responses given by the cells following preservation in the gelatin gels of the present invention are shown in
4. Protein synthesis measurements show a general cell function but do not indicate whether the latter is tissue specific. Following preservation using the gelatin gels of the present invention, cells were tested for their hormonal induction of the enzyme tyrosine aminotransferase. This is a liver specific property. It involves hormone receptor binding, occupied receptor translocation, transcription and translation. Typical results are shown in
5. In addition to maintaining liver specific functions, if the preserved cells are to approximate to the freshly isolated cells, they should not undergo phenotypic changes during preservation. The appearance of the enzyme gamma glutamyl transpeptidase is an early marker of liver cancer and is induced when hepatocytes are cultured at 37° C. (Edwards, Cancer Res., 42, 1107-1115, 1982).
6. Clearly “long term” phenotypic changes (such as the induction of γ-Glutamyl transpeptidase) are prevented. However, phenotypic changes occur at different rates. It was possible that rapid changes would still occur in the preservation systems. Possibly the earliest phenotypic change is the fall in cytochrome P450 which ultimately reduces the value of long-term cultures of hepatocytes in toxicity testing. The effect of various forms of culture on the levels of cytochrome P450 and the more stable cytochrome b5 were compared. As expected conventional culture led to a rapid decline in the level of cytochrome P450 (
7. As a further test of relevance to the in vivo situation, the isoenzyme profile of cytochrome P450 should not be changed by the preservation procedure.
Unlike cryopreservation, the cells are not in a state of suspended animation during preservation in the gelatin gels of the present invention. Some metabolism continues, although at a very low rate. To demonstrate that this was the case, a process, which could be monitored over a long time, was required. Protein synthesis measurements can only be measured over a relatively short time period due to the exhaustion on the labelling radioisotope and/or the turnover of newly synthesised proteins. However, protein degradation measurements, in the presence of the elevated levels of the unlabelled substance originally incorporated into the protein (to prevent reutilisation) can be made over an extended time. Measurements can be made both in terms of the loss of trichloroacetic acid insoluble material in the cells and the appearance of trichloroacetic acid soluble material in the medium.
Preservation using the gelatin gels of the present invention increased the half-life of the proteins by 20 fold. Under both conditions the radioactivity released from the trichloroacetic acid insoluble forms could be accounted for completely by the radioactivity recovered in the trichloroacetic acid soluble fraction. Cell death was therefore minimal under both sets of conditions. This was verified by total protein measurements, which showed that the cells were in a steady state of protein metabolism at 37° C. with only a marginal decrease in protein content during preservation. Protein degradation is an energy dependent process. During preservation the rate of protein degradation remains unchanged showing that detrimental effects do not occur over the measured time span.
Cell morphology is intrinsically linked to cell function. The inventors have performed studies which have demonstrated that changes to phenotype during culture at 37° C. are associated with changes to cell shape. Many other studies have also noted the importance of maintaining the in vivo cell shape if cell function is to be retained. See
For research purposes, it may be desirable to exposed cells to factors in order to study their effect on cell function e.g. cells are exposed to hormones to study induction profiles. If the cells are exposed prior to the study then the effect of the test agent could be invalid. With this in mind the effect of the various gelatin gel components, upon its preserving potential was performed with the potential aim of simplifying its make-up.
This study reveals the stability of the system, allowing a gel with a very defined composition to be developed.
Hepatocytes have many advantageous properties but at least one glaring difference from many cells in culture. In vitro, hepatocytes have a poor growth potential and the initially isolated cells cannot be maintained by subculturing. To assess the effect of the preservation systems on growing cells primary kidney proximal tubule cells have been prepared. The results show that the proximal tubule cells can be preserved using the gelatin gels of the present invention by using the same procedures. Marker enzymes of this cell type, such as alkaline phosphates and γ-Glutamyl transpeptidase are maintained at the level of the freshly isolated preparation for at least a week. The cells are maintained in preservation medium lacking bicarbonate. The viability of the isolated cells is show by trypan blue exclusion. Tubule viability is demonstrated by placing them in a hypotonic medium. Initially bleb formation is seen at the ends of the tubules but later occur along their length. Following the occurrence of blebs, the cells in the tubules begin to take up trypan blue. The preserved proximal tubule preparation shows one difference to that of hepatocytes. The proximal tubule cells will not attach to a support unless the gelatin is removed by washing the cells. Following cell attachment, at 37° C., the cells grow normally in bicarbonate-supplemented medium to form a confluent monolayer at the same rate as freshly isolated proximal tubule cells. The preservation procedures have no detrimental effects on cell growth. Data not shown.
The physicomechanical nature of the hydrolysed gelatin gel differs from that of the non-hydrolysed gelatin gel. The hydrolysed gelatin gel melts at a temperature lower than that of the non-hydrolysed gelatin gel. In addition the hydrolysed gelatin gel can be dispersed through the addition of an excess of aqueous medium (e.g. cell culture medium). The non-hydrolysed gelatin gel can only be dispersed using a heating step.
The hydrolysed gelatin gel is formed by the thermal treatment, under controlled conditions, of native gelatin. Changes to the molecular nature of the resulting gelatin is revealed by changes in the gelatin's resistance to precipitation by TCA (
Through experimentation it was found that hydrolysed gelatin inhibits hepatocyte attachment to synthetic cell culture substrata (in this case Primaria®).
SDS-PAGE analysis of gelatin, hydrolysed by varying degrees (see
These smaller polypeptides may account for the weaker gel, making it melt at a lower temperature. It also accounts for the susceptibility of the gel to dispersion by aqueous media. However more importantly it may also explain why a gel employing hydrolysed gelatin is superior for retaining cell viability and function to that of a preservation gel containing non-hydrolysed gelatin.
Hepatocytes have been preserved using preserving gelatin gels not only of varying compositions (see example 8) but also of differing configurations (i.e. relationship between the cells and the preserving gelatin gel) as shown in
The commercially available 75-bloom gelatin cannot be used for the preservation of hepatocytes since it is cytotoxic and causes great morphological abnormalities.
The use of autoclaved gelatin to preserve the cells provided identical results to that of preservation in 95° C. (2 h) treated gelatin.
A conclusion from
There is a progressive weakening of gel strength seen in out thermal treatments. The non-hydrolysed and 80° C. treated gelatin gel both require a heating step to release cells. In contrast both the 95° C. treated gel and autoclaved gelatin can be dissipated by aqueous medium or heating. In the same way that the 80° C. treated gelatin requires a shorter heating step than the non-hydrolysed gelatin to solubilise, the autoclaved gelatin gel is more readily dispersed by aqueous solutions while the 95° C. gel (2 h) is more robust and requires rimming prior to dispersion in aqueous medium.
To prove that such a temperature gradient does exist a 96-well microplate was used. Non-hydrolysed gelatin was added to each of the wells and the thermocouple could now be located precisely in an individual well.
All documents cited above are herein incorporated by reference.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0031065.6 | Dec 2000 | GB | national |
This application is a continuation application of Ser. No. 10/451,272 filed Oct. 14, 2003, which claims the benefit of co-pending PCT application PCT/GB01/05688 filed Dec. 20, 2001, which was published under PCT Article 21(2) in English on Jun. 27, 2002 (International Publication Number WO 02/49428 A1), which claims the benefit of GB 0031065.6 filed Dec. 20, 2000.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10451272 | Oct 2003 | US |
| Child | 12204955 | US |
