Patent Application: US-201514948272-A

Abstract:
it is not understood what causes or influences pattern formation in cells during the development of an organism . when animal / human cells are cultured in a petri dish the adherent cells attach to the bottom of the dish , whereas the non - adherent cells float in the growing medium . currently there are no specialized dishes for culturing non - adherent cells . we now show that non - adherent cells could be induced to form distinct patterns when cultured in an etched plastic dish . the non - adherent cells showed polarity when cultured in the etched plate . the polarity / pattern formation could be reversed with inhibitors specific for adhesion proteins . the phenomenon of pattern formation by non - adherent cells has wide applications in cell and developmental biology , diagnostics , microbiome research , biofluidics , drug discovery , industrial production of biological products , and also in biotechnology and bioengineering .

Description:
conventional methods of cell culture include seeding of cells on petri dishes . cell culture treated dishes are used to grow adherent cells , where they are attached to the bottom of the petri dish , whereas non - adherent cells do not attach to the dish . non - adherent cells ( including b cells , t cells , hybridomas ) are suspended in the medium ( schindler , 1969 ). most of the bacterial culture uses agar as a solid medium . the bacterial cells growing on the semi - solid agar form distinct colonies , which are later used for several studies . however , only less than 1 . 0 % of the bacteria are culturable . the ability to culture the majority of bacteria has impeded studies on new natural products and also has prevented factors that can contribute to both ecological balance and host health ( stewart , 2012 ). in vitro cell culture is the first step to test the efficacy of pharmacological drugs . there are no existing technologies for the culture of non - adherent cells thereby impeding studies on drugs targeting these cells . a technique to make the non - adherent cells adherent to a surface will lead to development of novel drugs that impact diseases like type i diabetes , arthritis , allergy etc . ( rello et al . 2005 ). in vitro culture is also used in the culture of organelles like choloroplast ( leiva - mora et al . 2010 ) for the synthesis of sugar or glucose for biofuel production . in vitro tissue culture is also used in the production of meat ( van eelan , 2007 ). the meat thus produced as an impact on the environment as lower amount of food and water are required to raise livestock . the carbon emission is also lowered when meat is cultured in vitro . recently 3d cell culture has gained popularity . a 3d cell culture is an artificially - created environment in which biological cells are permitted to grow or interact with their surroundings in all three dimensions . this is an improvement over the previous method of growing cells in 2d ( on a petri dish ) because the 3d model more accurately models the in vivo cells . these three - dimensional cultures are usually grown in bioreactors , small capsules in which the cells can grow into spheroids , or 3d cell colonies ( haycock , 2011 ; ravi et al . 2015 ). however , the 3d cultures are not suitable for non - adherent cells . cell culture in petri dishes is the first step in the production of novel biological products , or for identification of microorganisms , testing the efficacy of new pharmaceutical drugs , etc . as yet there are no in vitro systems for the culture of non - adherent cells . this led to the development of a new culture dish ( biosimulator ) that induces pattern formation in non - adherent cells . the method to design the biosimulator to induce pattern formation in non adherent cells is described herein . embodiments described herein demonstrate an in vitro system ( biosimulator ) for the culture of non - adherent cells . the in vitro system is an etched plastic surface . the non - adherent cells form distinct patterns after culture in the in vitro biosimulator . the non - adherent cells form distinct patterns based on the etching design . the present invention provides a mechanism by which non - adherent cells can form distinct patterns on modified plastic surfaces . the present invention provides mechanism by which the pattern formation of non - adherent cells could be altered . thus , in one aspect the present invention provides an efficacious mechanism to induce pattern formation in non - adherent cells . the present invention also provides a mechanism by which the non - adherent cells form polarity in in vitro culture . the non - adherent cells when cultured on an etched plate ( design : parallel lines ), adhere on top of the line on the upper part of the dish , whereas , the cells are attached below the line in the lower part of the dish . non - adherent cells of the present invention include cell and / or cell lines . examples of such cells and cell lines include primary cells ( e . g ., monocytes , t cell , b cell , rbc ) and / or cell lines / continuous cell lines such as hybridomas that are non - adherent . a cell culture may be grown in flasks , and subsequently passed to larger flasks to obtain larger volumes of material required to make cell lines . alternatively , the infected cell culture may be passed from flasks into subsequent roller bottles , spinner flasks , cell cubes , bioreactors , or any apparatus capable of growing cell culture on large scale in order to produce a suitable quantity of material . cell cultures may be frozen down in a suitable media and used for cell culture later . in certain aspects of the present invention , the 10 cm biosimulator is seeded with 200 , 000 to 400 , 000 cells . the cells are counted by a hemocytometer or any other electronic cell counter . the non - adherent cells form patterns 3 - 4 days after cell culture . the non - adherent entity could also be organelles like chloroplast , mitochondria , and ribosomes . the organelle culture could induce generation of bio - products like glucose , sugar , proteins , etc . the non - adherent entity could also be microorganisms including bacteria , virus fungi and parasites . only a small fraction of the microorganisms are culturable . the etches of a biosimulator facilitates growth of non - culturable microorganisms . the strategy could be used to identify microbiome of animals or for the culture of non - culturable animal and plant pathogens . in some embodiments , animal cells or cell lines including hybridomas ( in a biosimulator ) are typically grown at 37 ° c ., in the presence of 5 % co 2 . microorganisms can be cultured with or without co 2 , at varying temperatures . the pattern formation of non - adherence cells in a biosimulator can be observed by microscopy . the pattern formation of cells in a biosimulator can be observed with or without chemical dyes or stains . etches are made on the plastic surface with steel blades or lasers or any other material that could form the etching pattern . the etching pattern could also be modified using nano - materials like graphene . biofouling is an undesirable growth of microorganisms on probes or medical devices . currently biofouling is prevented by using specialized coatings . the present invention could predict the areas of the probes or devices susceptible to biofouling by etching the pattern on the biosimulator . based on the growth of the cells on a particular pattern , the probes or devices could be designed so that biofouling could be prevented . in cardiovascular diseases , the white blood cells ( wbcs ) including monocytes can block the arteries . culturing of wbcs from patients susceptible to cardiovascular diseases in a biosimulator could predict early diagnosis of the disease . the the malaria parasite , plasmodium resides in the red blood cells . recent studies by lu et al ( 2008 , 2010 ) demonstrated that rbcs are culturable . the biosimulator could be used to diagnose plasmodium infected rbcs . in certain embodiments the efficacy of pharmaceutical drugs on non - adherent cells could be studied using a biosimulator . those drugs that prevent adhesion of the cells can be identified . we had demonstrated that drugs that inhibit adhesin can prevent cell adhesion . new classes of drugs that blocks arteries could be identified using this invention . a 10 cm plastic cell culture dish was used to fabricate the biosimulator . a sterile sharp stainless steel blade was used for etching . etching was done in a laminar flow hood to maintain sterility . different patterns were etched on the plastic surface . primary cells or hybridomas were cultured at a concentration of 250 , 000 cells per 10 cm biosimulator . the biosimulator was incubated at 37 ° c ., with 5 % co2 . on the third day the non - adherent cells formed distinct patterns in the biosimulator . the non - adherent cells ( eg : hybridoma ) was treated with salicylic acid or the adhesion inhibitor pectasol ( which prevents cancer metastasis ) ( jiang et al . 2013 ). the non - adherent cells were cultured on an etched polystyrene biosimulator . there was no pattern formation on the first and second day of culture . after 3 days of culture the cells formed distinct pattern on the culture dish . all the cell lines tested ( the hybridomas 4b7 , 10d9 , 1a10 , 99d , sp2 / 0 , b56t ) formed distinct patterns on the etched plastic surface . the pattern formation corresponded to the etched line on the plastic surface . when the biosimulator had etched horizontal lines , the non - adherent cells were seen on top of the etched line , whereas , on the lower half of the dish the non - adherent cells were below the etched line . due to technical constraints to photograph a whole biosimulator under the microscope the cell alignment on the etched lines are shown graphically ( fig1 ). the cells were closely packed on the etched line . the experiment demonstrated that non - adherent cells could be converted to adherent cells and they could be induced to form distinct patterns on an etched surface . the pattern formation in non - adherent cells was found to be influenced by the etch design . cells were cultured on different etched designs . when the non - adherent cells were cultured on concentric squares / rectangles , the cells formed distinct patterns on the etched line . whereas , when small squares were etched in the biosimulator , the cells were adhered on two sides inside and two sides outside the square . when concentric circles were etched the cells were adhered on the circle in the upper part of the circle , whereas on the lower half of the circle the cells adhered inside the circle . similar patterns were also observed when small circles were etched on the edges of plastic dishes . when triangles were etched on edges of the dishes , the cells were always adhered to the inner two sides ( fig2 ). based on these studies we also observed that cells have affinity to different sides when etching different shapes like spiral structures ( figure not shown ). the phenomenon might be useful in designing probes for biomedical applications salicylic acid is known to prevent cell - cell interaction and is used in animal models of diabetes ( cao et al . 2012 ), but its mechanism of action is not clearly known . when salicylic acid was treated with the non - adherent cells they inhibited cell adhesion to the etched surface ( fig3 ). the adhesion inhibitor pectasol ( which prevents cancer metastasis ) was also used in our studies ( jiang et al . 2013 ). treatment of non - adherent cells with pectasol did not prevent cell proliferation , however , it prevented the cells to adhere to the plastic surface ( fig3 c ). the non - adherent cells lost the orientation property ; the cells were found floating in the medium and did not have any affinity for the etched surface . the in vitro experiments with drugs to inhibit adhesion demonstrated that the phenomenon of pattern formation could be employed in drug discovery studies . the study demonstrated that the non - adherent cells could be induced to form patterns in a biosimulator . cao y , dubois d c , almon r r , jusko w j . biopharm drug dispos . 2012 ; 33 : 285 - 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