A hybrid combination of in vitro cultured buccal mucosal cells using two different methodologies, complementing each other in successfully repairing a stricture-inflicted human male urethral epithelium

Horiguchi, A.; Kushibiki, T.; Yoshine, M.; Shinchi, M.; Ojima, K.; Hirano, Y.; Katoh, S.; Iwasaki, M.; Surya Prakash, V.; Ichiyama, K.; Senthilkumar, R.; Preethy, S.; Abraham, S. J.

doi:10.1101/2023.08.29.555240

bioRxiv · 10.1101/2023.08.29.555240

A hybrid combination of in vitro cultured buccal mucosal cells using two different methodologies, complementing each other in successfully repairing a stricture-inflicted human male urethral epithelium

Horiguchi, A.·Kushibiki, T.·Yoshine, M.·Shinchi, M.·Ojima, K.·Hirano, Y.·Katoh, S.·Iwasaki, M.·Surya Prakash, V.·Ichiyama, K.·Senthilkumar, R.·Preethy, S.·Abraham, S. J.

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Abstract

BackgroundAutologous buccal mucosal tissue derived cell transplantation techniques in repairing a stricture inflicted male urethral epithelium have been evolving. There was not much of clarity on the cell type, in vitro culture methods and the mode of transplantation, until we reported our buccal epithelium expanded and encapsulated in scaffold-hybrid approach to urethral stricture (BEES-HAUS) clinical study yielding a successful engraftment and repair with a long-term patency. We herein report with technical clarity on the advantages of mixing two-dimensional (2D) monolayer cultured fibroblast like cells and three dimensional (3D) thermo-reversible gelation polymer (TGP) cultured cells; the former secreting IGF-1, a cytokine known for its healing effects and the latter expressing epithelial surface markers in flow cytometry, both sourced from human buccal tissue, together transplanted using TGP as a carrier. MethodsHuman buccal tissues (n=22) redundant after urethroplasty surgery was used after informed consent and IEC approval. They were enzyme digested, divided into two portions; one was cultured as monolayer method (2D) and the other in 3D in TGP. Flowcytometry and quantification of IGF-1 in cell culture supernatant through the culture period were undertaken. ResultsIn flowcytometry, the cells on day 0, lacked AE1/AE3 - pancytokeratin expression indicative of epithelial phenotype of culture, which progressively increased in the 3D-TGP group, during invitro culture of up to 21 days. The 2D showed expression of only fibroblasts like cells that were negative for AE1/AE3 but positive for CD140b. IGF-1 secretion was significantly higher in 2D cultures than in 3D-TGP (p-value < 0.05). ConclusionThe 3D- TGP cultured cells of epithelial nature and the 2D cultured fibroblast like cells secreting IGF-1, together when transplanted using TGP scaffold as a carrier, adapted to a hostile in vivo milieu after releasing the fibrous strands with urethrotomy, successfully engrafted and repaired a stricture-inflicted male urethral epithelium in the BEES-HAUS procedure. While this hybrid combination of cells are considered to have potential in managing other epithelial damages, further research of such hybrid combination and their behaviour in disease affected environments may help to expand this solution in regenerating and repairing other tissues and organs as well. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/555240v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@1e00b7eorg.highwire.dtl.DTLVardef@12886aorg.highwire.dtl.DTLVardef@b9fbb3org.highwire.dtl.DTLVardef@3bd247_HPS_FORMAT_FIGEXP M_FIG C_FIG Illustration of pathogenesis of urethral stricture and the contribution of the hybrid combination of two-dimensional (2D) and three dimensional, 3D-TGP (Thermo-reversible gelation polymer) cultured cells to the successful repair of the stricture-inflicted male urethral epithelium in the buccal epithelium expanded and encapsulated in scaffold-hybrid approach to urethral stricture (BEES-HAUS) technique.

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Horiguchi, A., Kushibiki, T., Yoshine, M., Shinchi, M., Ojima, K., Hirano, Y., Katoh, S., Iwasaki, M., Surya Prakash, V., Ichiyama, K., Senthilkumar, R., Preethy, S., Abraham, S. J.. 2023-08-30. A hybrid combination of in vitro cultured buccal mucosal cells using two different methodologies, complementing each other in successfully repairing a stricture-inflicted human male urethral epithelium. https://doi.org/10.1101/2023.08.29.555240

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