1167: Towards an Autologous 3D Skin-like Tissue Harboring Patient-Derived Fibroblasts, Keratinocytes, T-cells and Macrophages

Sasha Shenk¹, Jonathan Garlick¹, Lev Brown², Jamie Riesenberg², Christian Evans², Julia Jaffe Zweifach², Andrew Macklin², Tamar Abel³, Noelle Kosarek⁴, Mengqi Huang⁵, Avi Smith¹, Tammara Wood³, Gretel Torres⁶, Patricia pioli⁶ and Michael Whitfield⁷, ¹Tufts University School of Dental Medicine, Boston, MA, ²Tufts University, Boston, MA, ³Dartmouth College, Hanover, NH, ⁴Dartmouth Geisel School of Medicine, Lebanon, NH, ⁵University of Pittsburgh, Pittsburgh, PN, ⁶Geisel School of Medicine at Dartmouth, Lebanon, NH, ⁷Dartmouth Geisel School of Medicine, Department of Biomedical Data Science, Lebanon, NH

Background/Purpose: Scleroderma (Systemic Sclerosis; SSc) is an autoimmune disease of unknown etiology that is characterized by vascular dysfunction, fibrosis, and inflammation. A lack of human autologous SSc disease models has created an unmet need for preclinical drug screening tissue models that can predict the success or failure of drugs designed to treat SSc. Recent advances in our lab have resulted in 3D skin-like tissue-based disease models that incorporate SSc patient-derived macrophages and fibroblasts. We now seek to develop 3D skin-like tissue models that additionally incorporate SSc patient-derived keratinocytes and T cells. We hope to more accurately recapitulate SSc patient subtypes that will be an important advancement over currently available 3D tissues.

Methods: Skin keratinocytes isolated from SSc and control patients that were tested using Colony Forming Efficiency (CFE) assays to determine their growth potential. CD4+ T cells were isolated from peripheral blood mononuclear cells (PBMCs) by magnetic bead isolation and tested for homogeneity using flow cytometry. 3D skin-like tissues (HSE= human skin equivalent) were constructed by seeding fibroblasts, keratinocytes and macrophages into a bovine collagen gel and grown at an air-liquid interface for 2 weeks. SSc-T-cells were then seeded onto a second tissue insert and HSEs were layered onto them. 3D skin-like tissues (Fig. 1) were constructed with varying combinations of SSc patient-derived cells that included: (1) fibroblasts; (2) keratinocytes with optimal proliferative potential, (3) CD4+ T cells and (4) macrophages. This created a 3D tissue that combined all four cell types from the same SSc patient to create a fully autologous, patient-specific 3D skin-like model of SSc.

Results: Morphologic analysis of the 3D tissue model showed that T-cells migrated into the tissue and remained viable as indicated by elevated levels of IL-6 only when T cells were present in the tissues (Fig. 1, D). CFE assays found that two SSc patient-derived keratinocyte strains (SF-15.2 and SF14.2) showed high CFEs of 11% and 18% that were sufficient for 3D tissue fabrication. When SSc patient-matched keratinocytes and fibroblasts were used to fabricate HSEs, these autologous tissues developed a more well-differentiated epithelium than in tissues with cells that were not autologous (Fig 2). We established that 3D tissues can incorporate 4 SSc-derived cell types that can recapitulate the cellular heterogeneity seen in SSc in vivo.

Conclusion: We have determined that that (1) fully autologous 3D skin-like tissues can be constructed with 4 different SSc patient-derived cell types, (2) SSc-derived tissues containing these multiple cells from the same person with SSc (autologous tissues) result in improved tissue morphology and phenotype when compared to tissues constructed with cells from different SSc patient skins, (3) these multiple cell types contribute to intercellular cross-talk needed to optimize skin tissue architecture to study SSc. These autologous 3D tissues will streamline screening of promising candidate drugs designed to improve treatment of SSc so they can safely and efficiently enter human clinical trials.
Figure 1. T cells in skin equivalents are functional- A) H&E image of 3D skin containing patient-derived fibroblasts, Macrophages and T cells. B) IHC staining for CD206-Macrophages (red) CD4-T cells (green) on bottom of tissue that migrated into dermis in 4 days (C). T cell function was shown by elevation of IL-6 secretion in T cell-containing tissues when compared to control or macrophage-only containing tissues (D).

Figure 2. Fabrication of fully autologous human tissues with SSc cells- HSE skin-like tissues were constructed with patient-matched, SSc-derived keratinocytes (SK15.2) and fibroblasts (SF15.2). Tissues were grown for 2 weeks revealing well-differentiated epithelium and well-structured dermis.
Disclosures: S. Shenk, None; J. Garlick, None; L. Brown, None; J. Riesenberg, None; C. Evans, None; J. Jaffe Zweifach, None; A. Macklin, None; T. Abel, None; N. Kosarek, None; M. Huang, None; A. Smith, None; T. Wood, None; G. Torres, None; P. pioli, None; M. Whitfield, Corbus Pharmaceuticals, Celdara Medical LLC, Bristol-Myers Squibb(BMS).