A kodecyte (ko•de•cyte) is a

living cell The cell is the basic structural and functional unit of life forms. Every cell consists of a cytoplasm enclosed within a membrane, and contains many biomolecules such as proteins, DNA and RNA, as well as many small molecules of nutrients an ...

that has been modified (koded) by the incorporation of one or more function-spacer-lipid constructs (FSL constructs) to gain a new or novel biological, chemical or technological function. The cell is modified by the lipid tail of the FSL construct incorporating into the bilipid membrane of the cell. All kodecytes retain their normal

vitality Vitality (, , ) is the capacity to live, grow, or develop. More simply it is the property of having life. The perception of vitality is regarded as a basic psychological drive and, in philosophy, a component to the will to live. As such, peopl ...

and functionality while gaining the new function of the inserted FSL constructs. The combination of dispersibility in

biocompatible Biocompatibility is related to the behavior of biomaterials in various contexts. The term refers to the ability of a material to perform with an appropriate host response in a specific situation. The ambiguity of the term reflects the ongoing de ...

media, spontaneous incorporation into cell membranes, and apparent low toxicity, makes FSL constructs suitable as research tools and for the development of new diagnostic and

therapeutic A therapy or medical treatment (often abbreviated tx, Tx, or Tx) is the attempted remediation of a health problem, usually following a medical diagnosis. As a rule, each therapy has indications and contraindications. There are many different ...

applications.

The technology

Structural analogy of a sunflower to space filling models of selected FSL constructs

Kode FSL constructs consist of three components; a functional

moiety Moiety may refer to: Chemistry * Moiety (chemistry), a part or functional group of a molecule ** Moiety conservation, conservation of a subgroup in a chemical species Anthropology * Moiety (kinship), either of two groups into which a society is ...

(F), a spacer (S) and a

lipid Lipids are a broad group of naturally-occurring molecules which includes fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, phospholipids, and others. The functions of lipids include ...

(L). Function groups on FSL constructs that can be used to create kodecytes include

saccharide In organic chemistry, a carbohydrate () is a biomolecule consisting of carbon (C), hydrogen (H) and oxygen (O) atoms, usually with a hydrogen–oxygen atom ratio of 2:1 (as in water) and thus with the empirical formula (where ''m'' may or may ...

s (including ABO blood group-related determinants,

sialic acid Sialic acids are a class of alpha-keto acid sugars with a nine-carbon backbone. The term "sialic acid" (from the Greek for saliva, - ''síalon'') was first introduced by Swedish biochemist Gunnar Blix in 1952. The most common member of this gr ...

s, hyaluronin polysaccharides),

fluorophore A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with se ...

biotin Biotin (or vitamin B7) is one of the B vitamins. It is involved in a wide range of metabolic processes, both in humans and in other organisms, primarily related to the utilization of fats, carbohydrates, and amino acids. The name ''biotin'', bor ...

, and a range of

peptides Peptides (, ) are short chains of amino acids linked by peptide bonds. Long chains of amino acids are called proteins. Chains of fewer than twenty amino acids are called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides. A p ...

. Although kodecytes are created by modifying natural cells, they are different from natural cells. For example, FSL constructs, influenced by the composition of the lipid tail, are laterally mobile in the membrane and some FSL constructs may also cluster due to the characteristics of the functional group (F). As FSL constructs are anchored in the membrane via a lipid tail (L) it is believed they do not participate in

signal In signal processing, a signal is a function that conveys information about a phenomenon. Any quantity that can vary over space or time can be used as a signal to share messages between observers. The ''IEEE Transactions on Signal Processing'' ...

transduction, but may be designed to act as agonists or antagonists of the initial binding event. FSL constructs will not actively pass through the plasma membrane but may enter the cell via membrane invagination and endocytosis. The "koding" of cells is stable (subject to the rate of turnover of the membrane components). FSL constructs will remain in the membrane of inactive cells (e.g. red blood cells) for the life of the cell provided it is stored in lipid free media. In the peripheral circulation FSL constructs are observed to be lost from red cell kodecytes at a rate of about 1% per hour. The initial "koding" dose and the minimum level required for detection determine how long the presence of "kodecytes" in the circulation can be monitored. For red blood "kodecytes" reliable monitoring of the presence of the "kodecytes" for up to 3 days post intravenous administration has been demonstrated in small mammals. The spacer (S) of a FSL construct has been selected so as to have negligible cross-reactivity with serum antibodies so kodecytes can be used with undiluted serum. By increasing the length of the FSL spacer from 1.9 to 7.2 nm it has been shown sensitivity can improve two-fold in red cell agglutination based kodecyte assays. However, increasing the size of the spacer further from 7.2 to 11.5 nm did not result in any further enhancement. Koded biomembrane

Technology Video

To view a simple video explaining how Kode Technology works, click the following link: https://www.youtube.com/watch?v=TIbjAl5KYpA

Methodology

Preparation of kodecytes and kodevirions

FSL constructs, when in solution ( saline) and in contact, will spontaneously incorporate into cell membranes. The methodology involves simply preparing a solution of FSL constructs in the range of 1–1000 μg/ mL, with the concentration used determining the amount of antigen present on the kodecyte. The ability to control antigen levels on the outside of a kodecyte has allowed for manufacture of quality control sensitivity systems and serologic teaching kits incorporating the entire range of serologic agglutination reactions. The actual concentration will depend on the construct and the quantity of construct required in the membrane. One part of FSL solution is added to one part of cells (up to 100%

suspension Suspension or suspended may refer to: Science and engineering * Suspension (topology), in mathematics * Suspension (dynamical systems), in mathematics * Suspension of a ring, in mathematics * Suspension (chemistry), small solid particles suspend ...

) and they are incubated at a set temperature within the range of 4–37 °C (39–99 °F) depending on temperature compatibility of the cells being modified. The higher the temperature, the faster the rate of FSL insertion into the membrane. For red blood cells incubation for 2 hours at 37 °C achieves >95% FSL insertion with at least 50% insertion being achieved within 20 minutes. In general, for carbohydrate based FSLs insertion into red blood cells, incubation for 4 hours at room temperature or 20 hours at 4 °C are similar to one hour at 37 °C . The resultant kodecytes do not required to be washed, however this option should be considered if an excess of FSL construct is used in the "koding process". Kodecytes can also be created ''

in vivo Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and ...

'' by injection of constructs directly into the circulation. However this process will modify all cells in contact with the constructs and usually require significantly more construct than ''in vitro'' preparation, as FSL constructs will preferentially associate with free lipids. The ''in vivo'' creation of kodecytes is untargeted and FSL constructs will insert into all cells non-specifically, but may show a preference for some cell types. Diagnostic serological analyses including

flow cytometry Flow cytometry (FC) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles. In this process, a sample containing cells or particles is suspended in a fluid and injected into the flo ...

and scanning electron microscopy usually can't see a difference between "kodecytes" and unmodified cells. However, when compared with natural cells there does appear to be a difference between

IgM Immunoglobulin M (IgM) is one of several isotypes of antibody (also known as immunoglobulin) that are produced by vertebrates. IgM is the largest antibody, and it is the first antibody to appear in the response to initial exposure to an antig ...

and

IgG Immunoglobulin G (Ig G) is a type of antibody. Representing approximately 75% of serum antibodies in humans, IgG is the most common type of antibody found in blood circulation. IgG molecules are created and released by plasma B cells. Each IgG ...

antibody reactivities when the functional group (F) is a monomeric peptide antigen. IgM antibodies appear to react poorly with kodecytes made with FSL peptides. Furthermore, FSL constructs may have a restricted antigen/epitope and may not react with a monoclonal antibody unless the FSL construct and monoclonal antibody are complementary. Kodecytes can be studied using standard histological techniques. Kodecytes can be fixed after "koding" subject to the functional moiety (F) of the FSL construct being compatible with the fixative. However, freeze cut or formalin-fixed freeze cut tissues are required because the lipid based FSL constructs (and other glycolipids) will be leached from the "kodecytes" in paraffin imbedded samples during the deparaffination steps.

Nomenclature

Koded membranes are described by the construct and the concentration of FSL (in μg/ mL) used to create them. For example, kodecytes created with a 100 μg/mL solution of FSL-A would be termed A100 kodecytes. If multiple FSL constructs were used then the definition is expanded accordingly, e.g. A100+B300 kodecytes are created with a solution containing 100 μg/mL solution of FSL-A and 300 μg/mL solution of FSL-B. The "+" symbol is used to separate the construct mixes, e.g. A100+B300. If FSL concentrations are constant then the μg/mL component of the terminology can be dropped, e.g. A kodecytes. Alternatively unrelated constructs such as FSL-A and FSL-biotin will create A+biotin kodecytes, etc. If different cells are used in the same study then inclusion of the cell type into the name is recommended, e.g. RBC A100 kodecytes vs WBC A100 kodecytes, or platelet A100 kodecytes, etc.

Applications

Kode Technology has been used for the ''

in vitro ''In vitro'' (meaning in glass, or ''in the glass'') studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called "test-tube experiments", these studies in biology an ...

'' modification of murine

embryo An embryo is an initial stage of development of a multicellular organism. In organisms that reproduce sexually, embryonic development is the part of the life cycle that begins just after fertilization of the female egg cell by the male spe ...

spermatozoa A spermatozoon (; also spelled spermatozoön; ; ) is a motile sperm cell, or moving form of the haploid cell that is the male gamete. A spermatozoon joins an ovum to form a zygote. (A zygote is a single cell, with a complete set of chromosomes, ...

zebra fish The zebrafish (''Danio rerio'') is a freshwater fish belonging to the minnow Family (biology), family (Cyprinidae) of the Order (biology), order Cypriniformes. Native to South Asia, it is a popular aquarium, aquarium fish, frequently sold under ...

epithelial Epithelium or epithelial tissue is one of the four basic types of animal tissue, along with connective tissue, muscle tissue and nervous tissue. It is a thin, continuous, protective layer of compactly packed cells with a little intercellula ...

endometrial The endometrium is the inner epithelial layer, along with its mucous membrane, of the mammalian uterus. It has a basal layer and a functional layer: the basal layer contains stem cells which regenerate the functional layer. The functional layer ...

cells and

red blood cell Red blood cells (RBCs), also referred to as red cells, red blood corpuscles (in humans or other animals not having nucleus in red blood cells), haematids, erythroid cells or erythrocytes (from Greek ''erythros'' for "red" and ''kytos'' for "holl ...

s to create cellular quality controls systems, serologic kits (teaching), rare

antigen In immunology, an antigen (Ag) is a molecule or molecular structure or any foreign particulate matter or a pollen grain that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune response. ...

expression, add infectious markers onto cells, modified cell adhesion/interaction/separation/immobilisation, and labelling. It has also been

intravascular The blood vessels are the components of the circulatory system that transport blood throughout the human body. These vessels transport blood cells, nutrients, and oxygen to the tissues of the body. They also take waste and carbon dioxide away f ...

ly infused for

modification of blood cells and neutralisation of circulating

antibodies An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...

and in ''in vivo'' imaging of circulating bone marrow kodecytes in zebrafish. Kode FSL constructs have also been applied to non-biological surfaces such as modified cellulose, paper, silica, polymers, natural fibers, glass and metals and has been shown to be ultra-fast in labelling these surfaces.

References

{{reflist, 30em, refs= Henry SM. Engineering the surface of red cells with synthetic glycolipids (KODETM CAE) to create ABO analytical sensitivity controls and xeno-modified cells. (invited lecture) 2nd International Symposium on ABO Incompatibility in Transplantation, Göteborg, Sweden, 2005 Xenotransplantation 2005; 12(5): 356 {{cite journal , last1 = Frame , first1 = Tom , last2 = Carroll , first2 = Tim , last3 = Korchagina , first3 = Elena , last4 = Bovin , first4 = Nicolai , last5 = Henry , first5 = Stephen , title = Synthetic glycolipid modification of red blood cell membranes , volume = 47 , issue = 5 , pages = 876–882 , year = 2007 , doi = 10.1111/j.1537-2995.2007.01204.x , journal = Transfusion , pmid = 17465953 , citeseerx = 10.1.1.494.2776 , s2cid = 18086433 {{cite journal , last1 = Hult , first1 = Annika K , last2 = Frame , first2 = Tim , last3 = Chesla , first3 = Scott , last4 = Henry , first4 = Stephen , last5 = Olsson , first5 = Martin L , title = Flow cytometry evaluation of red blood cells mimicking naturally-occurring ABO subgroups following modification with variable amounts of FSL-A and B constructs , volume = 52 , issue = 2 , pages = 247–251 , year = 2012 , doi = 10.1111/j.1537-2995.2011.03268.x , journal = Transfusion , pmid = 21812783 , s2cid = 5984970 {{cite journal , last1 = Flower , first1 = R , last2 = Lin P-H , first2 = Heathcote D , last3 = Chan , first3 = M , last4 = Teo , first4 = D , last5 = Selkirk , first5 = A , last6 = Shepherd , first6 = R , last7 = Henry , first7 = S , year = 2008 , title = Insertion of KODE peptide constructs into red cell membranes: Creating artificial variant MNS blood group antigens. ISBT Regional Congress, Macao SAR China, 2008". (P-396) , journal = Vox Sanguinis , volume = 95 , issue = Suppl 1, pages = 203–204 {{cite journal , last1 = Heathcote , first1 = D , last2 = Flower , first2 = R , last3 = Henry , first3 = S , year = 2008 , title = Development of novel alloantibody screening cells – the first example of the addition of peptide antigens to human red cells using KODE technology. ISBT Regional Congress, Macao SAR China, 2008". (P-303) , journal = Vox Sanguinis , volume = 95 , issue = Suppl 1, page = 174 {{cite journal , last1 = Henry , first1 = Stephen M , title = Modification of red blood cells for laboratory quality control use , volume = 16 , issue = 6 , pages = 467–472 , year = 2009 , doi = 10.1097/MOH.0b013e328331257e , journal = Current Opinion in Hematology , pmid = 19680123 , s2cid = 37416831 {{cite journal , last1 = Heathcote , first1 = Damien , last2 = Carrol , first2 = Tim , last3 = Wang , first3 = Jui-Jen , last4 = Flower , first4 = Robert , last5 = Rodionov , first5 = Igor , last6 = Tuzikov , first6 = Alexander , last7 = Bovin , first7 = Nicolai , last8 = Henry , first8 = Stephen , title = Novel antibody screening cells, MUT+Mur kodecytes, created by attaching peptides onto erythrocytes , volume = 50 , issue = 3 , pages = 635–641 , year = 2010 , doi = 10.1111/j.1537-2995.2009.02480.x , journal = Transfusion , pmid = 19912581 , s2cid = 20952307 {{cite journal , last1 = Chesla , first1 = S , last2 = Henry , first2 = S , last3 = Eatz , first3 = R , last4 = Sinor , first4 = L , title = Solid phase syphilis test utilizing KODE technology , volume = 50 , pages = 196A–197A , year = 2010 , doi = 10.1111/j.1537-2995.2010.02833_1.x , pmid = 20815863 , journal = Transfusion , s2cid = 222195124 {{cite journal, url=http://carbohyd.siobc.ras.ru/printable.php?id=381, vauthors=Komarraju S, Chesla S, Bovin N, Henry S , title= Syphilis-kodecytes – novel function-spacer-lipid (FSL) modified red cells capable of sensitive and specific detection of syphilis antibodies, year=2010, journal= FEBS Journal , volume=277 , issue= S1, pages=97–98, doi=10.1111/j.1742-4658.2010.07680.x, pmc=7164047 , hdl=10292/2142 , hdl-access=free {{cite journal , vauthors=Blake D, Lan A, Love D, Bovin N, Henry S , title=Fluorophore-kodecytes – fluorescent function-spacer-lipid (FSL) modified cells for in vitro and in vivo analyses, doi=10.1111/j.1742-4658.2010.07680.x , journal=FEBS Journal , issue=1 , year=2010 , pages=37–271 , volume=277 , pmc=7164047 , hdl=10292/2142, hdl-access=free {{cite journal , last1 = Nadarajan , first1 = V.S. , last2 = Laing , first2 = A. A. , last3 = Saad , first3 = S. M. , last4 = Usin , first4 = M , title = Prevalence and specificity of red-blood-cell antibodies in a multiethnic South and East Asian patient population and influence of using novel MUT+Mur+ kodecytes on its detection , volume = 102 , issue = 1 , pages = 65–71 , year = 2011 , doi = 10.1111/j.1423-0410.2011.01507.x , journal = Vox Sanguinis , pmid = 21592136 , s2cid = 20297050 {{cite journal , last1 = Henry , first1 = Stephen , last2 = Komarraju , first2 = Sarvani , last3 = Heathcote , first3 = Damien , last4 = Rodinov , first4 = Igor L , title = Designing peptide-based FSL constructs to create Miltenberger kodecytes , volume = 6 , issue = 2 , pages = 306–312 , year = 2011 , doi = 10.1111/j.1751-2824.2011.01505.x , journal = ISBT Science Series , s2cid = 82441272 , doi-access = free {{cite journal , last1 = Georgakopoulos , first1 = T , last2 = Komarraju , first2 = Sarvani , last3 = Henry , first3 = Stephen , last4 = Bertolini , first4 = Joseph , title = An improved Fc function assay utilising CMV antigen coated red blood cells generated with synthetic Function-Spacer-Lipid constructs , volume = 102 , issue = 1 , pages = 72–78 , year = 2011 , doi = 10.1111/j.1423-0410.2011.01512.x , journal = Vox Sanguinis , pmid = 21749406 , s2cid = 9758322 {{cite journal , last1 = Oliver , first1 = Caroline , last2 = Blake , first2 = Debbie , last3 = Henry , first3 = Stephen , title = In vivo neutralization of anti-A and successful transfusion of A antigen incompatible red cells in an animal model , volume = 51 , issue = 12 , pages = 2664–2675 , year = 2011 , doi = 10.1111/j.1537-2995.2011.03184.x , journal = Transfusion , pmid = 21599675 , s2cid = 205724219 {{cite journal , last1 = Oliver , first1 = Caroline , last2 = Blake , first2 = Debbie , last3 = Henry , first3 = Stephen , title = Modeling transfusion reactions and predicting in vivo cell survival with kodecytes , volume = 51 , issue = 8 , pages = 1723–1730 , year = 2011 , doi = 10.1111/j.1537-2995.2010.03034.x , journal = Transfusion , pmid = 21303367 , s2cid = 24736518 {{cite journal , last1 = Blake , first1 = Debbie A , last2 = Bovin , first2 = Nicolai V , last3 = Bess , first3 = Dan , last4 = Henry , first4 = Stephen M , title = FSL Constructs: A Simple Method for Modifying Cell/Virion Surfaces with a Range of Biological Markers Without Affecting their Viability , volume = 54 , issue = e3289 , year = 2011 , doi = 10.3791/3289 , pmid = 21847082 , pmc = 3211133 , journal = Journal of Visualized Experiments {{cite journal , last1 = Lan , first1 = C-C , last2 = Blake , first2 = D , last3 = Henry , first3 = S , last4 = Love , first4 = D R , title = Fluorescent Function-Spacer-Lipid construct labelling allows for real-time in vivo imaging of cell migration and behaviour in zebrafish (Danio rerio) , year = 2012 , doi = 10.1007/s10895-012-1043-3 , journal = Journal of Fluorescence , pmid=22434405 , volume=22 , issue=4 , pages=1055–63 , s2cid = 14406691 {{cite journal , last1 = Korchagina , first1 = Elena , last2 = Tuzikov , first2 = Alexander , last3 = Formanovsky , first3 = Andrey , last4 = Popova , first4 = Inna , last5 = Henry , first5 = Stephen , last6 = Bovin , first6 = Nicolai , title = Toward creating cell membrane glycolandscapes with glycan lipid constructs , year = 2012 , doi = 10.1016/j.carres.2012.03.044 , journal = Carbohydrate Research , pmid=22551471 , volume=356 , pages=238–46 {{cite book , last1 = Henry , first1 = Stephen , last2 = Rodionov , first2 = Igor , title = FSL-RFG(Maleimide) FSL Construction Kit Technical Bulletin , publisher = Scholarly Commons , year = 2012 , hdl = 10292/2241 {{cite book , last1 = Henry , first1 = Stephen , last2 = Perry , first2 = Holly , title = FSL-A+B(tri) Serologic Teaching Kit Technical Bulletin , publisher = Scholarly Commons , year = 2012 , hdl = 10292/2827 {{cite journal , last1 = Henry , first1 = Stephen , last2 = Barr , first2 = Katie , last3 = Oliver , first3 = Caroline , title = Modeling transfusion reactions with kodecytes and enabling ABO incompatible transfusion with Function-Spacer-Lipid constructs , publisher = ISBT Science Series , type = In Press

External links

Kodeycte.com

How Kode Technology works

Applications of kodecytes

CSL Limited, CSL application of kodecytes Biochemistry Biotechnology Laboratory techniques Molecular biology techniques Protein methods Nanotechnology