Patent Application: US-90744301-A

Abstract:
a process of using a fish plasma component as a nutrient medium component for tissue culture includes obtaining blood from a fish , separating plasma from the blood , and extracting one or more specific components of the plasma . the tissue is cultured using the extracted plasma components , and none of any remainder of the plasma , in a nutrient medium . the tissue cultured using the extracted plasma component is other than fish tissue , such as mammalian tissue or insect tissue .

Description:
the process begins with the consistent and reproducible conditions under which donor fish are reared . all fish used as plasma sources preferably are progeny of domesticated broodstock , inspected for fish disease according to the american fisheries society “ blue book ” standards , sexually immature , in the log - phase of growth , larger than two kilograms , reared by standard husbandry methods , and fed a commercially pelleted food appropriate to the species . water temperature at the time of bleeding is preferably 4 ° c . to 12 ° c . the fish are preferably starved for five days before bleeding to reduce proteolytic enzymes and non - protein nitrogen . each fish is stunned by a blow to the head , or by immersion in ice - water , or in water containing co 2 or other fish anesthetic , in order to stun the fish to a level of loss of reflex activity ( unconsciousness ) as defined by schreck and moyle , ( 1990 ). whole blood is then drawn from the caudal artery or vein with a sterile needle and syringe or vacuum tube containing an anticoagulant such as acd ( acid citrate dextrose ), trisodium citrate , or other anticoagulant commonly used in human blood - banking . whole blood is held for no more than four hours at 2 - 4 ° c ., and then centrifuged at 2 - 4 ° c . because of the large amounts of highly unsaturated fatty acids , plasma to be used for lipid extraction is handled under argon , or an antioxidant such as alphatocopherol , bht , or mercaptoethanol at less than 1 ppm is added . plasma is then frozen at − 80 ° c . for plasma lipids , an extraction procedure ( for example , that described in detail by condie , 1979 , or ando , 1996 ) is applied to whole plasma . in summary , this process utilizes fumed silica to adsorb the lipids from the plasma fraction . lipids are then eluted from the silica with sodium citrate at ph 10 - 11 and dialyzed against a saline solution , and additional antioxidants ( for example , ascorbic acid , bha , bht ) are added . the lipid is then analyzed for cholesterol content and concentrated to a level of 5 to 15 mgs / ml cholesterol . the lipid is then stored under vacuum or argon at − 80 ° c . for fibrinogen extraction and purification , the method of silver et al ., 1995 is used . this method is based on ammonium sulfate precipitations , which yields greater than 95 % pure fibrinogen ( by sds - page ). thrombin is prepared by the method of ngai and chang , 1991 . these extraction techniques are illustrative of those currently in use , but other techniques may be equally effective . the essential requirements are that all process temperatures must remain below 4 ° c ., there must be no cytotoxic chemical residues in the product , and plasma lipids must be protected from oxidation . a green monkey kidney cell line ( vero ) commonly used in commercial culture , the promega nonradioactive cell proliferation assay ( fisher healthcare , houston , tex . ), and serum - free media , vp - sfm ( life technologies , inc ., grand island , n . y . ), were used to evaluate the fish lipid component . the frozen fish lipid was thawed in a water bath at 2 - 4 ° c . assays were conducted using 24 - well polystyrene culture plates . each well was seeded with 30 , 000 cells in vp - sfm medium containing 5 % fetal calf serum ( fbs ). the cells were allowed to attach and spread for a 24 - hour period , and then the growth medium was removed by aspiration . all wells were rinsed thoroughly with the vp - sfm medium and the test formulations ( 3 wells each ) were added . the cells were then incubated at 37 ° c . for 48 hours in a 5 % co 2 atmosphere in 95 % relative humidity . after 48 hours , the cultures were examined and quantified using the promega nonradioactive cell proliferation assay . this assay measures viable cells only and is based on a standard curve of cell concentrations determined for each cell type . results for each condition were averaged and statistically compared using anova ( one - way analysis of variance ). there was no significant difference between the number of viable cells in the vp - sfm and the vp - sfm plus the lower concentration of salmon lipid , showing that the fish material was not toxic . however , addition of salmon plasma lipid at the higher concentration to the media ( vp - sfm plus 1 . 0 mgs / l cholesterol ) enhanced growth significantly ( p =& lt ; 0 . 001 ). the highest concentration of salmon lipid ( 5 . 0 mgs / l ) was less effective ( fig3 ). these results show that the salmon plasma lipids enhance the growth of a mammalian cell line ( vero ) in culture . growing mammalian neurons in a gel made from fish plasma components is an example of in vitro cell culture with potential in vivo ( tissue engineering ) applications . cell survival and neurite process extension in gels are established models for nerve regeneration in vivo ( schense et al ., 2000 ). primary spinal cord neuronal cultures were prepared as described by dunham ( 1988 ) from embryos harvested from timed - pregnant female mice ( c57bl / 6j ; jackson laboratory , bar harbor , me .). culture media and conditions for the neurons were also as described by dunham ( 1988 ). lyophilized salmon fibrinogen and thrombin were reconstituted in water at room temperature , and the gels were prepared by treating 3 mg / l salmon fibrinogen with 1 . 5 u / ml salmon thrombin and adding 1 . 4 mm calcium in cell culture media . similar gels were prepared using lyophilized human and bovine fibrinogen and thrombin . in order to embed neurons in the gel , fibrinogen , neurons , and cell culture media were mixed together , and then thrombin was added . the solution was mixed gently 2 - 3 times and transferred to a polylysine - coated coverslip . the formation of the fist fibrin gels was similar to gels formed from mammalian material and resulted in a solid gel within 30 minutes at room temperature with a shear modulus of about 550 dynes / cm . after at least 30 minutes , the gels were covered with neuronal cell culture media and placed in a 37 ° c . cell culture incubator the neurons in the fish and mammalian fibrin gels were viewed on a nikon diaphot 300 inverted microscope , and images were captured with a micromax cooled ccd camera driven by inovision image processing software on a sgi 02 computer . images were processed and compiled using adobe photoshop 5 . 0 . neurite length was quantified using nih image , and all data was analyzed using kaleidagraph . after 2 days in culture , human fibrin gels began to disintegrate , and by day 4 , the gel was completely digested away , leaving only sparse cells attached to the glass . in contrast , bovine and fish gels remained intact for at least a week . fig4 and 5 show several examples of neuronal cell bodies ( arrowheads ) and extended processes ( arrows ) in the gels . fish fibrin gels contained multiple neurons with processes longer than those of the neurons in the bovine gels , and extending in three dimensions into the gel . quantitation of neurite length ( microns ) in fish gels compared to that in bovine gels reveals that neurite length in fish gels is greater by a factor of 2 . 3 ( fish gels = 416 . 25 ± 89 . 9 sem , n = 10 cells : bovine gels = 179 . 18 ± 20 . 9 sem , n = 8 cells ) ( fig6 ). these results show a clear and significant enhancement of neurite length for mammalian spinal cord neurons when they are cultured in a salmon fibrin gel instead of the mammalian gel . although only two cell types and three plasma components were tested , our experiments demonstrate that those with ordinary skill in the field of tissue culture may substitute salmonid plasma components for the mammalian plasma substances now used for mammalian tissue culture , and realize significant advantages from the fish material that were not provided by fish whole plasma and serum products . preferred and alternative embodiments have not been described in detail . it is contemplated , however , that various modifications of the disclosed embodiments fall within the spirit and scope of the invention . the scope of the appended claims , therefore should be interpreted to include such modifications , and is not limited to the particular embodiments disclosed herein . for example , the use of these and other fish plasma components in mammalian tissue culture , or fish plasma components in insect cell culture , especially in the production of recombinant proteins , is a contemplated aspect of the present invention to satisfy the same objects and provide the same advantages as those for mammalian cell culture . ando , s . 1996 . adsorption of serum lipoproteins from rainbow trout to dextran sulfate cellulose . fisheries science 62 ( 3 ): 410 - 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