M. Isani1, X. Hou1, A. Fode1, C. Schlieve1, T. C. Grikscheit1 1Children’s Hospital Los Angeles,Los Angeles, CA, USA
Introduction:
Splenectomy leaves patients susceptible to overwhelming post-splenectomy infection (OPSI), which results in high mortality. The spleen is remarkably regenerative as demonstrated by the phenomenon of splenosis that was originally noted after autotransplantation, necrosis, and then growth of splenic tissue in the peritoneum of patients after traumatic splenic rupture. Although splenosis sometimes generates functional spleen, it is built on a construct of necrotic tissue and has variable protection against OPSI. We hypothesized that omental implantation of human multicellular clusters of spleen stem/progenitor cells or splenic units (SU) on a biodegradable polymer scaffold could generate controlled functional tissue-engineered human spleen.
Methods:
After appropriate IACUC and IBC approval, human spleen samples from patients undergoing splenectomy were washed with Hank’s balanced salt solution (HBSS), and mechanically and chemically digested into SU in a variation of a murine protocol we have previously described. SU were loaded onto a 2mm biodegradable polyglycolic/poly-L lactic acid scaffold and implanted either into the omentum or subcutaneously in NOD/SCID mice. After four weeks, implants were harvested, stained with hemotoxylin and eosin, and immunostained with a specific human anti-lamin antibody, a nuclear envelope marker and anti-PCNA, proliferating cell nuclear antigen.
Results:
Omental implants generated visible human tissue-engineered spleen in mice (n=3). H&E staining revealed evidence of splenic structural elements. Immunofluorescence staining for human anti-lamin antibody confirmed that cells in TES derive from the human donor cells. Furthermore, the PCNA costain identified actively proliferating human cells (nuclear stain DAPI in blue, Figure 1A, anti-lamin in green, Figure 1B, anti-PCNA in pink, Figure 1C, and Figure 1D is a merged image with inset showing proliferating human cell, all images at 40x with 50μm scale bar).
Conclusion:
These experiments demonstrate growth of human donor cells into tissue-engineered spleen from splenic units implanted onto a biodegradable construct in mice. Additionally, proliferation of these proven human cells is recorded. Proliferation is fundamental to establishing, growing, and maintaining tissue-engineered organs. This approach may therefore prove beneficial in future protection against OPSI via direct implantation of human splenic units in a trauma setting or to control splenic regeneration in failed partial splenectomy.
