Patent Application: US-201214235909-A

Abstract:
the present invention aims to generate a fibrin gel for cell proliferation and transport using the patient &# 39 ; s own blood or compatible blood . the gel - preparation process consists in taking a quantity of blood from the patient , using sodium citrate as anticoagulant , separating off the citrated plasma by centrifugation , and resuspending the cells to be transported in the plasma . cacl 2 is added to the resulting suspension to form the gel , and the gel is cultured in an incubator to promote cell growth . the invention will be of use in cell growth for the purposes of implantation in cutaneous , cartilaginous , gingival and bone lesions , inter alias , and in the field of tissue engineering .

Description:
the technical issue raised in this application consists of providing a product and system preferably autologous allowing immobilize , proliferate and conveyed cells within a matrix for the purpose of tissue regeneration . the search of biomaterials that cell growth is a fertile ground . required materials that mimic the extracellular cell with its three - dimensional features . within these biomaterials collagen , gelatin , alginate and other materials have been used . among these , the use of fibrin has shown extraordinary properties to promote the growth of skin cells . however , in the clinical application of these cultures for the treatment of skin lesions is with the disadvantage that only clinical grade materials should be used . the latter exist and are called tissucol ® and beriplast ® in the clinic is used as fibrin sealants in different surgical procedures . found these products maintain the properties of cell growth promoting detected with the combination of thrombin and fibrinogen level in vitro research . however the above the cost of these products , given the complex microbiological analysis that require , makes them incompatible with the possibility of creating a commercial product containing fibrin , and cover large areas of damaged skin . materials that are safe for human beings , free of animal components and risk of transmission of known and emerging diseases are also required . a product of autologous origin will always be more secure from the microbiological standpoint than one heterologous , since although this polluted , will return to the same host . on the other hand , from the immunological point of view , antibodies and other proteins present in it will be those already existing in the individual , eliminating the possibility of immune rejection . the present invention is directed to generate a fibrin gel for proliferation and cell conduction from the patient &# 39 ; s own blood or blood compatible . the blood rate of 200 μl per cm of implant to prepare is taken using sodium citrate as an anticoagulant in a reason of 0 . 09 g per ml of blood . plasma citrated is separated by centrifugation . the cells are then suspended again in this plasma . to form the gel either as isolated clot inside a porous matrix is to add a cacl2 solution . the product thus obtained is of much lower cost than commercial “ fibrin glue ” or “ fibrin sealant ” and for being an autologous product , the risk of infection and immune rejection is avoided , and although it may be difficult to obtain large volumes of blood from a patient , this can be overcome using compatible blood . this system has been used to treat skin lesions with positive results . the application will be for purposes of cell growth in skin lesions implants , cartilage , osseous and gingival lesions among others . it can be anticipated that any application in the field of tissue engineering can use this technology . the novelty lies in the incorporation of the cells into a fibrin clot using plasma instead of purified fibrin and thrombin . the fibrin glues contain both components and some others from blood , but they are no equivalent to plasma since they are purified . also they incorporate additives , such as protease inhibitors that are not present in plasma . all shown references are ways for generating the fibrin clot , but not ways for integrating cells in a fibrin clot with purposes of tissue engineering . there are many publications that support the potential of growing cells in fibrin clots . for this , commercial fibrinogen and thrombin are acquired or commercial preparations known as fibrin glues are used , the most popular being tissucol ( baxter ) or beriplast ( csl behring ). these products allow the cells to be suspended in thrombin and then fibrinogen is added for making a cell containing clot . the problem is that for doing this with clinical and commercial purposes a lot of money should be expended because the products of clinical grade are very expensive . it is also difficult to be 100 % sure that the clot will not transmit any virus or unknown microorganism for which there are no screening tests with appropriate sensitivity . thus , the idea of generating fibrin for cell growth from the patient &# 39 ; s own blood to form the gel , either as an isolated clot or within a porous matrix , arises . according to the above , the product generated in the present invention has as main advantages that it has much lower cost than the commercial “ fibrin glues ” and for being and autologous product , it eliminates the risk of infection and immune rejection . on the other hand , the disadvantage that it can be difficult to obtain large volumes of blood from a compromised patient , is solved using compatible blood . for the support of the invention , a number of clinical trials have been developed in which the cells are incorporate into a porous matrix by the use of autologous blood . this system has been used to treat skin lesions with positive results . an object of the present invention is the application for the purpose of cell growth in implants for skin , cartilage , gingival and bone lesions , among others . the patent literature shows homologous products that differ of the invention in fundamental aspects . a description of the most relevant known in patents follows : beretta & amp ; grippi in the patent applications numbers us 2009258056 and wo 2007021344 and beretta & amp ; lodi in patent number u . s . pat . no . 6 , 368 , 298 , developed methods for regenerating tissue in a living organism , which include contacting the affected area with a net of solid fibrin containing platelets that release growth factors . the gel is a - cellular and contains platelets derived from autologous blood . similarly , baugh & amp ; lim in patent applications numbers us 2005152886 and us 2002159985 , and in the patent number u . s . pat . no . 6 , 444 , 228 , and hirsch & amp ; johnston in patent number ep0820314 , describe methods for obtaining autologous fibrin sealant , which does not contain incorporated cells but only platelets . the patent number u . s . pat . no . 5 , 185 , 001 by galanakis d ., discloses a kit which contains all the elements to produce a fibrin clot from autologous plasma , in order to use it to induce hemostasis ( clotting in a wound ), but no reports the inclusion of cells in it . moreover , in the chilean patent application cl 1439 - 2002 , obtaining of a dermal substitute from plasma , using calcium chloride during gelation is described , but just as same as previously disclosed inventions , the cell component from a culture is not incorporated . it is mentioned that it allows the invasion by cells and blood vessels , but it refers to those of the wound bed and not exogenously provided . additionally , there is a significant amount of scientific articles , as well as some patents already mentioned , pursuing the research of the same technical problem . however , they are all ways to generate the fibrin clot and not ways for integrating the cells into a fibrin clot with tissue engineering purposes . the examples below indicated , are incorporated by way of illustration only , to facilitate understanding of the invention , and not meant to limit in any way the scope of the requested claims . human fibroblasts are obtained from foreskins of surgeries due to phimosis in children under 6 years . the biopsy is washed in a sterile petri dish with 5 ml of phosphate buffered saline solution , 0 . 1 m ph 7 . 4 ( pbs ) ( gibco ®), containing a mixture of penicillin / streptomycin antibiotics 100 u / ml / 100 invitrogen ®) and amphotericin b 250 μg / ml ( fungizone ®, invitrogen ). to obtain separate fibroblasts and keratinocytes , the sample is incubated in trypsin 0 . 5 %, 5 . 3 mm edta ( gibco ®) or 30 minutes at 37 ° c . in incubator thermo forma ®. subsequently , using sterile forceps , the dermis and the epidermis are mechanically separated . the dermis is treated for 20 min with collagenase type i ( invitrogen ®) 2 mg / ml at 37 ° c . then , the obtained cell suspension is centrifuged and the precipitated obtained is washed with pbs to remove the enzyme . its cell viability is determined by stain exclusion with trypan blue . this is accomplished by microscopic examination of an aliquot of cell suspension mixed with trypan blue stain in a 1 : 1 ratio . then this suspension is placed in a neubauer chamber and viable cells are counted by observation in inverted microscope lieder ®. human fibroblasts obtained after enzymatic digestion , are re - suspended in eagle culture medium and modified by dulbecco ( dmem , gibco ®) supplemented with 10 % of fetal bovine serum (( invitrogen ®), 50 % ham f12 medium ( gibco ®) and 10 ml / ml biomyc 1 ( biological industries ®) in culture flasks t - 25 ( falcon ®), that are incubated in an incubator thermoformed , in a humidified atmosphere with 5 % co 2 and a temperature of 37 ° c . 50 % medium f12 of ham ( gibco ®) and 10 μl \ ml of biomyc 1 ( biological industries ®) in culture flasks t - 25 ( falcone ), that are incubated in an incubator thermoforma , in a humidified atmosphere with 5 % of co , and a temperature of 37 ° c . obtaining the plasma . the plasma is separated from whole blood collected into tubes containing sodium citrate at 3 . 2 %, centrifuged at 3000 rpm for 5 minutes . the separated plasma in sterile conditions under bio - safety hood , stored at − 20 ° c . until use . clotting test by visual observation : initial tests of clotting , are performed in microcentrifuge tubes by testing various concentrations of calcium , named formulations ( f ). for this , a constant volume of citrated plasma 100 μl is used with different volumes of dilutions of cacl 2 , to obtain different final concentrations of the same , generating 30 formulations as illustrated in table 2 : after preparing the 30 formulations , these are incubated with the constant volume of plasma at 37 ° c . for 20 minutes . they are visually evaluated , whether is coagulation in each formulation . furthermore , each clot is removed from the microcentrifuge tube with forceps , for evaluation of its mechanical strength , and thus the best 6 formulations are selected , based on the existence of clot and the greatest mechanical strength . for the determination of the clotting time a clot timer ( bbl fibrosystem ) is used , which allows to determine , more precisely , the time in which the clot forms . this test is performed only in the 6 preselected formulations in the preliminary stage . the stability of the clot is determined in 6 clots of the formulations selected with culture medium ( dmem , gibco ®), supplemented with 10 % of fetal bovine serum ( invitrogen ®), 50 % medium f12 of ham ( gibco ®). subsequently , it is incubated at 37 ° c . the degree of disintegration is evaluated visually on day 3 ( fig2 ). preparation of base polymer : for the base polymer , the following solutions are prepared : gelatin 1 % w / v , chitosan 2 % w / v in acetic acid 1 % v / v , and hyaluronic acid 0 . 01 % w / v according to the following protocol : the gelatin solution with chitosan and hyaluronic acid is mixed , and homogenized for 30 minutes with magnetic stirring at 50 ° c . subsequently , the mixture is poured into petri dishes that are refrigerated at 4 ° c . until the formation of gel . then , the gel is frozen slowly at − 20 ° c . for 8 hours . then , the polymer is carried to − 80 ° c . for 8 hours . the polymer is slowly immersed in liquid nitrogen for 3 minutes . finally , it is lyophilized for 48 hours in liobras lt01 lyophilizer . the lyophilisate is immersed in mes 50 mm ( 2 - morpholinoethane sulphonic acid ), for 30 minutes . then , this solution is discarded and reticulant solution composed by mes 50 mm , edc 30 mm ( 1 - ethyl -[ 3 , 3 - dimethylaminopropyl ] carbodiimide ) and nhs 8 mm ( n - hydroxysuccinimide ) are added . it is left to react for 2 hours . once frozen , it is immersed in liquid nitrogen for 3 minutes . for this experiment , the technique is mtt [( 3 - 4 , 5 - dimethylthiazolyl - 2 )- 2 , 5 - diphenyltetrazolium bromide ] was used , which allows quantification of viable cells . this technique is based on the reduction of mtt , only by those metabolically active cells , in a compound called “ formazan ” a soluble purple blue crystal , which can be quantified by spectrophotometry at 540 nm . mtt assay : obtaining fibroblasts to this test is performed as follows : culture flasks t - 75 ( falcon ®) with fibroblasts are washed with pbs , and then treated with trypsin . the cell suspension is placed in a 50 ml disposable centrifuge tube , and centrifuged at 3000 rpm for 5 minutes . then , the precipitate obtained is re - suspended in order to verify the number of viable cells . once realized the cell counting , the suspension is centrifuged again , and a portion of the cells are re - suspended in culture medium ( dmem , gibco ®), supplemented with 10 % of fetal bovine serum ( invitrogen ®), 50 % of medium f12 of ham ( gibco ®) and a mixture of penicillin / streptomycin antibiotics ( 100 u / ml / 100 μg / ml ), invitrogen ®), and the other in plasma . this assay is performed in 96 flat bottom wells ( falcon ®), where the proliferation of fibroblasts is evaluated , using different surfaces for its cultivation . the assay is performed from day 0 to day 3 , i . e . at 0 , 24 , 48 and 72 hours , and each condition is evaluated in triplicate with its level control for each day . also , only the two best previously selected formulations are considered for testing . detailed below is the mtt assay to examine the proliferation of fibroblasts on different surfaces : monolayer : human fibroblasts re - suspended in medium ( 5 × 10 6 cells per well ), are cultured directly in the well with 150 μl of culture medium . polymer matrix : the polymer matrix is placed in each well and the re - suspended human fibroblast are delivered in medium ( 5 × 10 6 cells per well ). culture medium is added until complete 150 μl . clot f17 : 30 μl of plasma with humans fibroblasts are deposited ( 5 × 10 6 cells per well ), and to this 45 μl of cacl 2 30 mm is added . is incubated at 37 ° c . until the clot formation and culture medium is then added until complete 150 μl . clot f27 : 30 μl of plasma with humans fibroblasts are deposited ( 5 × 10 6 cells per well ), and to this 22 . 5 μl of cacl2 50 mm is added . is incubated at 37 ° c . until the clot formation , and the culture medium is then added until complete 150 μl . su with f17 : on the polymer matrix , 30 μl of plasma with human fibroblasts ( 5 × 10 6 cells per well ) and 45 μl of cacl 2 30 mm are deposited . is incubated at 37 ° c . until clot formation , and then the culture medium is added until complete 150 μl . su with f27 : on the polymeric matrix , 30 μl of plasma with human fibroblasts ( 5 × 10 6 cells per well ), and 22 . 5 μl of 50 cacl 2 mm are deposited . is incubated at 37 ° c . until clot formation , and then the culture medium is added until complete 150 μl . mtt procedure : once assembled the experiment with the different cell culture conditions , mtt assay for day 0 ( 0 hours ) is performed to establish values of cell viability at the time of the beginning of the experiment . this procedure is subsequently repeated after 24 , 48 and 72 hours . the procedure consists of : add 50 μl of mtt solution to each well , corresponding to the evaluation day and incubate at 37 ° c . for 4 hours . subsequently , each solution in the wells is removed and transferred into the corresponding microcentrifuge tubes for each sample . 150 μl of trypsin 10 % is added to each well . the plate is again lead to the incubator at 37 ° c . for 1 hour . finally , 300 μl of lysis buffer are added into each well ( for the monolayer 400 μl ), vigorously pipetted , and the solution is transferred to each microcentrifuge tube ( this , in two steps , first add 100 μl or 200 μl , as appropriate , and in a second step add the remaining 200 μl ). the tubes are maintained at − 20 ° c . until photometric quantification . to do this , microfuge tubes with samples of each day are thawed . carefully , particle - free supernatants from each tube are extracted and 200 μl of each sample are deposited into a 96 wells plate . finally , the absorbance is read on plate reader elisa sensiscan ( merk ®) at 540 nm . 1 . system and method for preparing autologous fibrin glue . inventors : beretta roberto ; grippi nicholas a applicant : beretta roberto ; grippi nicholas a . publication info : jp2010115507 ( a )— 2010 may 27 , priority date : 2002 jan . 15 . 2 . systems and methods for preparing autologous fibrin . inventor : beretta roberto [ it ]; grippi nicholas a [ us ] applicant : cascade medical entpr llc [ us ]. publication info : us2009203613 ( a1 )— 2009 aug . 13 . priority date : 1997 jun . 24 . 3 . systems and methods for preparing autologous fibrin glue . inventor : beretta roberto [ it ]; grippi nicholas a [ us ]. applicant : cascade medical entpr llc [ us ]. publication info : us2009258056 ( a1 )— 2009 oct . 15 . priority date : 1997 jun . 24 . 4 . systems and methods for preparing autologous fibrin glue . inventor : beretta roberto [ it ]; grippi nicholas a [ us ]. applicant : cascade medical entpr llc [ us ]; beretta roberto [ it ] (+ 1 ). publication info : wo2007021344 ( a1 )— 2007 feb . 22 . priority date : 2005 aug . 17 . 5 . autologous fibrin sealant and method for making the same . inventor : baugh robert f [ us ]; lim lisa m [ us ] (+ 2 ) applicant : medtronic inc . publication info : us2005152886 ( a1 )— 2005 jul . 14 , u . s . pat . no . 7 , 811 , 607 ( b2 )— 2010 oct . 12 . priority date : 1996 apr . 30 . 6 . autologous fibrin sealant and method for making the same . inventor : baugh robert f [ us ]; lim lisa m [ us ] (+ 2 ) applicant : baugh robert f ,; lim lisa m , (+ 3 ). publication info : us2002159985 ( a1 )— 2002 oct . 31 , u . s . pat . no . 6 , 830 , 762 ( b2 )— 2004 dec . 14 priority date : 1996 apr . 30 . 7 . preparing autologous fibrin glue . inventor : beretta roberto [ it ]; lodi sergio [ it ]. applicant : beretta roberto [ it ]. publication info : u . s . pat . no . 6 , 368 , 298 ( b1 )— 2002 apr . 9 . priority date : 1997 jun . 24 . 8 . single use system for preparing autologous plasma and fibrin gel . inventor : whitmore elaine [ us ]. applicant : whitmore elaine . publication info : u . s . pat . no . 6 , 197 , 194 ( b1 )— 2001 mar . 6 . priority date : 1995 mar . 24 . 9 . autologous fibrin sealant and method for making the same . inventor : baugh robert f [ us ]; lim lisa m [ us ] (+ 2 ) applicant : medtronic inc [ us ]. publication info : u . s . pat . no . 6 , 444 , 228 ( b1 )— 2002 sep . 3 . priority date : 1996 apr . 30 . 10 . preparation of autologous plasma and fibrin gel . inventor : whitmore elaine [ us ]. applicant : johnson & amp ; johnson medical [ us ]. publication info : u . s . pat . no . 5 , 935 , 437 ( a )— 1999 aug . 10 . priority date : 1995 mar . 24 . 11 . process of preparation of an autologous fibrin glue . inventor : tarantino flavio [ it ]; benincasa carlo [ it ] (+ 2 ) applicant : tarantino flavio [ it ]; benincasa carlo [ it ] (+ 2 ) publication info : w0981 1925 ( a1 )— 1998 mar . 26 . priority date : 1996 sep . 18 . 12 . autologous fibrin glue and methods for its preparation and use . inventor : hirsh jack [ ca ]; johnston marilyn [ ca ] (+ 1 ). applicant : hamilton civic hospitals res [ ca ]. publication info : ep0820314 ( a1 )— 1998 jan . 28 . priority date : 1995 apr . 6 . 13 . method of preparing autologous plasma fibrin and application apparatus therefore . inventor : galanakis dennis k [ us ]. applicant : univ new york state res found [ us ]. publication info : u . s . pat . no . 5 , 185 , 001 ( a )— 1993 feb . 9 . 14 . metodo of obtencion of sustituido dermico a partir of fibrina . s , cl 200201439 . 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( 1 ) hair , ( 2 ) sweat glands , ( 3 ) basal lamina , ( 4 ) blood vessels ( hib , 2001 ) fig2 . histology and immunohistochemistry of microencapsulated cells , a . a cell in a homogeneous area of fibrin stained with methylene blue ( microencapsulation day ); b . cell in metaphase located close to the fibrin which is stained with erythrosin ( 48 hours after microencapsulation ); c . three cells embedded in the fibrin gel ( 24 hours after microencapsulation d . a pair of cells located in the pores of the fibrin matrix ( 48 hours after microencapsulation ); e . group of cells located within the fibrin gel ( 96 hours after microencapsulation ); f . expansion of the cells cluster of e ( arrows ) where a cell in metaphase is observed ; from g . to i . : immunohistochemistry of a cell cluster growing in a fibrin gel ; g . control with arteta trichrome staining ; h . immunolocalization of cytokeratin and i . immunolocalization of vimentin ( acevedo c . et al , bioprocess biosyst eng ., 32 ( 3 ): 341 - 51 , 2009 ). fig3 . study of selected clots stability ( f17 , f22 , f23 , f27 ). panel a : appearance at the beginning . panel b : appearance at three days post - incubation at 37 ° c . fig4 . growth curves of human fibroblasts , grown on the mentioned surfaces . statistically significant differences in growth with respect to day 0 are quoted (*) ( anova , p & lt ; 0 . 05 ).