{{short description|Modified living cell}} {{Multiple issues| {{primary sources|date=June 2011}} {{COI|date=June 2011}} }}
A '''kodecyte''' (ko•de•cyte) is a living cell that has been modified (koded) by the incorporation of one or more function-spacer-lipid constructs (FSL constructs)<ref name="Korchagina2012" /><ref name="Henry2009" /><ref name=":0">{{Cite journal|title = Synthetic glycolipid-like constructs as tools for glycobiology research, diagnostics, and as potential therapeutics|journal = Biochemistry (Moscow)|date = 2015-07-16|issn = 0006-2979|pages = 857–871|volume = 80|issue = 7|doi = 10.1134/S0006297915070068|pmid = 26542000|first1 = E. Y.|last1 = Korchagina|first2 = S. M.|last2 = Henry|s2cid = 14965044}}</ref> 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 and functionality while gaining the new function of the inserted FSL constructs. The combination of dispersibility in biocompatible 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 applications.
==The technology== [[File:Structural analogy of a sunflower to space filling models of selected FSL constructs.png|thumb|400x400px|Structural analogy of a sunflower to space filling models of selected FSL constructs. The two FSL constructs on the left are FSL-peptides based on partially carboxymethylated oligoglycine (CMG2) spacers with 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) lipids. The 3rd and 4th constructs are FSL-biotin based on CMG but with DOPE and sterol (δ-oxycarbonylaminovaleric acid derivative of cholesterol) lipids, respectively. The final FSL construct is a typical trisaccharide, conjugated via an O(CH<sub>2</sub>)<sub>3</sub>NH spacer to an activated adipate derivative of the diacyl lipid DOPE.]]
Kode FSL constructs consist of three components;<ref name=":0" /><ref name="Frame2007" /> a functional moiety (F), a spacer (S) and a lipid (L).
Function groups on FSL constructs that can be used to create kodecytes include saccharides (including ABO blood group-related determinants,<ref name="Frame2007" /><ref name="Hult2008" /><ref name="Henry2005b" /> sialic acids, hyaluronin polysaccharides), fluorophores,<ref name="Blake2010" /><ref name=":2">{{Cite journal|title = Incorporation of fluorescein conjugated function-spacer-lipid constructs into the red blood cell membrane facilitates detection of labeled cells for the duration of ex-vivo storage|journal = Journal of Immunological Methods|doi = 10.1016/j.jim.2016.01.003|pmid = 26773455|first1 = Katrina K.|last1 = Ki|first2 = Robert L.|last2 = Flower|first3 = Helen M.|last3 = Faddy|first4 = Melinda M.|last4 = Dean|date = Jan 7, 2016|volume=429|pages=66–70|url = http://espace.library.uq.edu.au/view/UQ:379199/UQ379199_OA.pdf}}</ref> biotin,<ref name="Oliver2011b" /> and a range of peptides.<ref name="Heathcote2010" /><ref name="Heathcote2008" /><ref name="Flower2008" /><ref name="Chesla2010" /><ref name="Komarraju2010" /><ref name="Nadarajan2011" /><ref name="Henry2012a" /><ref name="Henry2011" /><ref name="Georgakopoulos2011" />{{further|topic=FSL functional groups|Function-Spacer-Lipid_construct#Functional_Groups}}
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).<ref name="Korchagina2012" /> As FSL constructs are anchored in the membrane via a lipid tail (L) it is believed they do not participate in signal 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.<ref name="Blake2010" />
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.<ref name="Blake2010" /> In the peripheral circulation FSL constructs are observed to be lost from red cell kodecytes at a rate of about 1% per hour.<ref name="Oliver2011b" /><ref name="Oliver2011a" /> 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.<ref name="Oliver2011b" />
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.<ref name="Korchagina2012" />[[File:Koded biomembrane.svg|thumb|400x400px|A plasma membrane modified with FSL constructs (by analogy to sunflower), creating a kodecyte membrane.]]
=== Technology Video === To view a simple video explaining how Kode Technology works, click the following link: https://www.youtube.com/watch?v=TIbjAl5KYpA
== Methodology ==
thumb|400x400px|Preparation of kodecytes. Simply mix cells with an FSL solution (containing 1 or more FSLs) and incubate for 10–120 minutes at 37 °C (or at temperatures as low as 4 °C). The constructs will spontaneously incorporate into the membrane and no further steps are required.
FSL constructs, when in solution (saline) and in contact, will spontaneously incorporate into cell membranes.<ref name="Blake2011" /> 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<ref name="Henry2009" /> and serologic teaching kits incorporating the entire range of serologic agglutination reactions.<ref name="Henry2012b" /> 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) and they are incubated at a set temperature within the range of {{convert|4|–|37|C|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.<ref name="Blake2011" /> 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'' by injection of constructs directly into the circulation.<ref name="Oliver2011a" /> 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.<ref name="Oliver2011a" /> 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<ref name="Frame2007" /> including flow cytometry<ref name="Hult2008" /> 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 and 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.<ref name="Heathcote2010" /><ref name="Henry2011" /> 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.<ref name="Heathcote2010" /><ref name="Henry2011" />
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.<ref name="Blake2011" />
== Nomenclature ==
Koded membranes are described by the construct and the concentration of FSL (in μg/mL) used to create them.<ref name="Blake2011" /> 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'' modification of murine embryos, spermatozoa, zebra fish, epithelial/endometrial cells and red blood cells<ref name=":0" /><ref name="Frame2007" /><ref name="Hult2008" /><ref name=":2" /><ref name="Heathcote2008" /><ref name="Flower2008" /><ref name=":1">{{Cite journal|title = Mapping the fine specificity of ABO monoclonal reagents with A and B type-specific function-spacer-lipid constructs in kodecytes and inkjet printed on paper|journal = Transfusion|date = 2014-10-01|issn = 1537-2995|pages = 2477–2484|volume = 54|issue = 10|doi = 10.1111/trf.12661|first1 = Katie|last1 = Barr|first2 = Elena|last2 = Korchagina|first3 = Ivan|last3 = Ryzhov|first4 = Nicolai|last4 = Bovin|first5 = Stephen|last5 = Henry|pmid=24749871|s2cid = 206336530}}</ref> to create cellular quality controls systems,<ref name="Henry2009" /><ref name=":0" /><ref name="Heathcote2010" /> serologic kits (teaching),<ref name="Henry2012b" /><ref>{{Cite journal|title = Training students in serologic reaction grading increased perceptions of self-efficacy and ability to recognize serologic reactions but decreased grading accuracy|journal = Transfusion|date = 2015-06-01|issn = 1537-2995|pages = 1572–1579|volume = 55|issue = 6pt2|doi = 10.1111/trf.12985|first1 = Holly|last1 = Perry|first2 = Stephen|last2 = Henry|pmid=25564758|s2cid = 10378319}}</ref> rare antigen expression, add infectious markers onto cells,<ref name=":0" /><ref name="Chesla2010" /><ref name="Georgakopoulos2011" /> modified cell adhesion/interaction/separation/immobilisation,<ref name=":0" /><ref name="Blake2010" /><ref name="Oliver2011b" /> and labelling.<ref name="Hult2008" /><ref name=":2" /> It has also been intravascularly infused for in vivo modification of blood cells and neutralisation of circulating antibodies<ref name=":0" /><ref name="Oliver2011a" /><ref name="Henry2012c" /> and in ''in vivo'' imaging of circulating bone marrow kodecytes in zebrafish.<ref name="Lan2012" /> Kode FSL constructs have also been applied to non-biological surfaces such as modified cellulose, paper,<ref name=":1" /> silica, polymers, natural fibers, glass and metals and has been shown to be ultra-fast in labelling these surfaces.<ref name=":0" /><ref name="Williams 118">{{Cite journal|title = Ultra-Fast Glyco-Coating of Non-Biological Surfaces|journal = International Journal of Molecular Sciences|date = 2016-01-16|pages = 118|volume = 17|issue = 1|doi = 10.3390/ijms17010118|pmid = 26784187|pmc = 4730359|first1 = Eleanor|last1 = Williams|first2 = Katie|last2 = Barr|first3 = Elena|last3 = Korchagina|first4 = Alexander|last4 = Tuzikov|first5 = Stephen|last5 = Henry|first6 = Nicolai|last6 = Bovin| doi-access=free }}</ref>
==See also== *Function-Spacer-Lipid construct *Kodevirion
==References== {{reflist|30em| refs=
<ref name="Henry2005b">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</ref>
<ref name="Frame2007"> {{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 }} </ref>
<ref name="Hult2008"> {{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 }} </ref>
<ref name="Flower2008">{{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 }}</ref>
<ref name="Heathcote2008">{{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 }}</ref>
<ref name="Henry2009"> {{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 }} </ref>
<ref name="Heathcote2010"> {{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 }} </ref>
<ref name="Chesla2010"> {{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 }} </ref>
<ref name="Komarraju2010">{{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}}</ref>
<ref name="Blake2010"> {{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}}</ref>
<ref name="Nadarajan2011"> {{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 | doi-access = free }} </ref>
<ref name="Henry2011"> {{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 }} </ref>
<ref name="Georgakopoulos2011"> {{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 }} </ref>
<ref name="Oliver2011a"> {{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 }} </ref>
<ref name="Oliver2011b"> {{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 }} </ref>
<ref name="Blake2011"> {{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 }} </ref>
<ref name="Lan2012"> {{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 | hdl = 10292/3475 | hdl-access = free }} </ref>
<ref name="Korchagina2012"> {{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 }}</ref>
<ref name="Henry2012a"> {{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 }} </ref>
<ref name="Henry2012b"> {{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 }} </ref>
<ref name="Henry2012c"> {{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 | journal = ISBT Science Series | date = 2012 | volume = 7 |issue=1 | pages = 106–111 | doi = 10.1111/j.1751-2824.2012.01563.x }} </ref>
<!--oliver 2011a becomes Oliver2011b--> }}
==External links== *[http://www.kodecyte.com] Kodeycte.com *[https://www.youtube.com/watch?v=G9jjTJ5Pt4k] How Kode Technology works *[https://www.youtube.com/watch?v=l3KLkFN6dYQ] Applications of kodecytes *[https://www.youtube.com/watch?v=SK1VPW7jt9c] CSL application of kodecytes
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