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)[1][2][3] 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

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(CH2)3NH spacer to an activated adipate derivative of the diacyl lipid DOPE.

Kode FSL constructs consist of three components;[3][4] 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,[4][5][6] sialic acids, hyaluronin polysaccharides), fluorophores,[7][8] biotin,[9] and a range of peptides.[10][11][12][13][14][15][16][17][18]

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).[1] 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.[7]

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.[7] In the peripheral circulation FSL constructs are observed to be lost from red cell kodecytes at a rate of about 1% per hour.[9][19] 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.[9]

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.[1]

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

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.[20] 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[2] and serologic teaching kits incorporating the entire range of serologic agglutination reactions.[21] 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 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 .[20] 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.[19] 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.[19] 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[4] including flow cytometry[5] 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.[10][17] 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.[10][17]

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.[20]

Nomenclature

Koded membranes are described by the construct and the concentration of FSL (in μg/mL) used to create them.[20] 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[3][4][5][8][11][12][22] to create cellular quality controls systems,[2][3][10] serologic kits (teaching),[21][23] rare antigen expression, add infectious markers onto cells,[3][13][18] modified cell adhesion/interaction/separation/immobilisation,[3][7][9] and labelling.[5][8] It has also been intravascularly infused for in vivo modification of blood cells and neutralisation of circulating antibodies[3][19][24] and in in vivo imaging of circulating bone marrow kodecytes in zebrafish.[25] Kode FSL constructs have also been applied to non-biological surfaces such as modified cellulose, paper,[22] silica, polymers, natural fibers, glass and metals and has been shown to be ultra-fast in labelling these surfaces.[3][26]

See also

References

  1. 1 2 3 Korchagina, Elena; Tuzikov, Alexander; Formanovsky, Andrey; Popova, Inna; Henry, Stephen; Bovin, Nicolai (2012). "Toward creating cell membrane glycolandscapes with glycan lipid constructs". Carbohydrate Research. 356: 238–46. doi:10.1016/j.carres.2012.03.044. PMID 22551471.
  2. 1 2 3 Henry, Stephen M (2009). "Modification of red blood cells for laboratory quality control use". Current Opinion in Hematology. 16 (6): 467–472. doi:10.1097/MOH.0b013e328331257e. PMID 19680123. S2CID 37416831.
  3. 1 2 3 4 5 6 7 8 Korchagina, E. Y.; Henry, S. M. (2015-07-16). "Synthetic glycolipid-like constructs as tools for glycobiology research, diagnostics, and as potential therapeutics". Biochemistry (Moscow). 80 (7): 857–871. doi:10.1134/S0006297915070068. ISSN 0006-2979. PMID 26542000. S2CID 14965044.
  4. 1 2 3 4 Frame, Tom; Carroll, Tim; Korchagina, Elena; Bovin, Nicolai; Henry, Stephen (2007). "Synthetic glycolipid modification of red blood cell membranes". Transfusion. 47 (5): 876–882. CiteSeerX 10.1.1.494.2776. doi:10.1111/j.1537-2995.2007.01204.x. PMID 17465953. S2CID 18086433.
  5. 1 2 3 4 Hult, Annika K; Frame, Tim; Chesla, Scott; Henry, Stephen; Olsson, Martin L (2012). "Flow cytometry evaluation of red blood cells mimicking naturally-occurring ABO subgroups following modification with variable amounts of FSL-A and B constructs". Transfusion. 52 (2): 247–251. doi:10.1111/j.1537-2995.2011.03268.x. PMID 21812783. S2CID 5984970.
  6. ↑ 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
  7. 1 2 3 4 Blake D, Lan A, Love D, Bovin N, Henry S (2010). "Fluorophore-kodecytes – fluorescent function-spacer-lipid (FSL) modified cells for in vitro and in vivo analyses". FEBS Journal. 277 (1): 37–271. doi:10.1111/j.1742-4658.2010.07680.x. hdl:10292/2142. PMC 7164047.
  8. 1 2 3 Ki, Katrina K.; Flower, Robert L.; Faddy, Helen M.; Dean, Melinda M. (Jan 7, 2016). "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" (PDF). Journal of Immunological Methods. 429: 66–70. doi:10.1016/j.jim.2016.01.003. PMID 26773455.
  9. 1 2 3 4 Oliver, Caroline; Blake, Debbie; Henry, Stephen (2011). "Modeling transfusion reactions and predicting in vivo cell survival with kodecytes". Transfusion. 51 (8): 1723–1730. doi:10.1111/j.1537-2995.2010.03034.x. PMID 21303367. S2CID 24736518.
  10. 1 2 3 4 Heathcote, Damien; Carrol, Tim; Wang, Jui-Jen; Flower, Robert; Rodionov, Igor; Tuzikov, Alexander; Bovin, Nicolai; Henry, Stephen (2010). "Novel antibody screening cells, MUT+Mur kodecytes, created by attaching peptides onto erythrocytes". Transfusion. 50 (3): 635–641. doi:10.1111/j.1537-2995.2009.02480.x. PMID 19912581. S2CID 20952307.
  11. 1 2 Heathcote, D; Flower, R; Henry, S (2008). "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)". Vox Sanguinis. 95 (Suppl 1): 174.
  12. 1 2 Flower, R; Lin P-H, Heathcote D; Chan, M; Teo, D; Selkirk, A; Shepherd, R; Henry, S (2008). "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)". Vox Sanguinis. 95 (Suppl 1): 203–204.
  13. 1 2 Chesla, S; Henry, S; Eatz, R; Sinor, L (2010). "Solid phase syphilis test utilizing KODE technology". Transfusion. 50: 196A–197A. doi:10.1111/j.1537-2995.2010.02833_1.x. PMID 20815863. S2CID 222195124.
  14. ↑ Komarraju S, Chesla S, Bovin N, Henry S (2010). "Syphilis-kodecytes – novel function-spacer-lipid (FSL) modified red cells capable of sensitive and specific detection of syphilis antibodies". FEBS Journal. 277 (S1): 97–98. doi:10.1111/j.1742-4658.2010.07680.x. hdl:10292/2142. PMC 7164047.
  15. ↑ Nadarajan, V.S.; Laing, A. A.; Saad, S. M.; Usin, M (2011). "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". Vox Sanguinis. 102 (1): 65–71. doi:10.1111/j.1423-0410.2011.01507.x. PMID 21592136. S2CID 20297050.
  16. ↑ Henry, Stephen; Rodionov, Igor (2012). FSL-RFG(Maleimide) FSL Construction Kit Technical Bulletin. Scholarly Commons. hdl:10292/2241.
  17. 1 2 3 Henry, Stephen; Komarraju, Sarvani; Heathcote, Damien; Rodinov, Igor L (2011). "Designing peptide-based FSL constructs to create Miltenberger kodecytes". ISBT Science Series. 6 (2): 306–312. doi:10.1111/j.1751-2824.2011.01505.x. S2CID 82441272.
  18. 1 2 Georgakopoulos, T; Komarraju, Sarvani; Henry, Stephen; Bertolini, Joseph (2011). "An improved Fc function assay utilising CMV antigen coated red blood cells generated with synthetic Function-Spacer-Lipid constructs". Vox Sanguinis. 102 (1): 72–78. doi:10.1111/j.1423-0410.2011.01512.x. PMID 21749406. S2CID 9758322.
  19. 1 2 3 4 Oliver, Caroline; Blake, Debbie; Henry, Stephen (2011). "In vivo neutralization of anti-A and successful transfusion of A antigen incompatible red cells in an animal model". Transfusion. 51 (12): 2664–2675. doi:10.1111/j.1537-2995.2011.03184.x. PMID 21599675. S2CID 205724219.
  20. 1 2 3 4 Blake, Debbie A; Bovin, Nicolai V; Bess, Dan; Henry, Stephen M (2011). "FSL Constructs: A Simple Method for Modifying Cell/Virion Surfaces with a Range of Biological Markers Without Affecting their Viability". Journal of Visualized Experiments. 54 (e3289). doi:10.3791/3289. PMC 3211133. PMID 21847082.
  21. 1 2 Henry, Stephen; Perry, Holly (2012). FSL-A+B(tri) Serologic Teaching Kit Technical Bulletin. Scholarly Commons. hdl:10292/2827.
  22. 1 2 Barr, Katie; Korchagina, Elena; Ryzhov, Ivan; Bovin, Nicolai; Henry, Stephen (2014-10-01). "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". Transfusion. 54 (10): 2477–2484. doi:10.1111/trf.12661. ISSN 1537-2995. PMID 24749871. S2CID 206336530.
  23. ↑ Perry, Holly; Henry, Stephen (2015-06-01). "Training students in serologic reaction grading increased perceptions of self-efficacy and ability to recognize serologic reactions but decreased grading accuracy". Transfusion. 55 (6pt2): 1572–1579. doi:10.1111/trf.12985. ISSN 1537-2995. PMID 25564758. S2CID 10378319.
  24. ↑ Henry, Stephen; Barr, Katie; Oliver, Caroline. "Modeling transfusion reactions with kodecytes and enabling ABO incompatible transfusion with Function-Spacer-Lipid constructs" (In Press). ISBT Science Series. {{cite journal}}: Cite journal requires |journal= (help)
  25. ↑ Lan, C-C; Blake, D; Henry, S; Love, D R (2012). "Fluorescent Function-Spacer-Lipid construct labelling allows for real-time in vivo imaging of cell migration and behaviour in zebrafish (Danio rerio)". Journal of Fluorescence. 22 (4): 1055–63. doi:10.1007/s10895-012-1043-3. PMID 22434405. S2CID 14406691.
  26. ↑ Williams, Eleanor; Barr, Katie; Korchagina, Elena; Tuzikov, Alexander; Henry, Stephen; Bovin, Nicolai (2016-01-16). "Ultra-Fast Glyco-Coating of Non-Biological Surfaces". International Journal of Molecular Sciences. 17 (1): 118. doi:10.3390/ijms17010118. PMC 4730359. PMID 26784187.
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