Cell sheet technology: Influence of culture conditions on in vitro‐cultivated corneal stromal tissue for regenerative therapies of the ocular surface

Abstract

The in vitro reconstruction of stromal tissue by long‐term cultivation of corneal fibroblasts is a smart approach for regenerative therapies of ocular surface diseases. However, systematic investigations evaluating optimized cultivation protocols for the realization of a biomaterial are lacking. This study investigated the influence of supplements to the culture media of human corneal fibroblasts on the formation of a cell sheet consisting of cells and extracellular matrix. Among the supplements studied are vitamin C, fetal bovine serum, L‐glutamine, components of collagen such as L‐proline, L‐4‐hydroxyproline and glycine, and TGF‐β1, bFGF, IGF‐2, PDGF‐BB and insulin. After long‐term cultivation, the proliferation, collagen and glycosaminoglycan content and light transmission of the cell sheets were examined. Biomechanical properties were investigated by tensile tests and the ultrastructure was characterized by electron microscopy, small‐angle X‐ray scattering, antibody staining and ELISA. The synthesis of extracellular matrix was significantly increased by cultivation with insulin or TGF‐β1, each with vitamin C. The sheets exhibited a high transparency and suitable material properties. The production of a transparent, scaffold‐free, potentially autologous, in vitro‐generated construct by culturing fibroblasts with extracellular matrix synthesis‐stimulating supplements represents a promising approach for a biomaterial that can be used for ocular surface reconstruction in slowly progressing diseases.

Isolation and Propagation of Human Corneal Stromal Keratocytes for Tissue Engineering and Cell Therapy

“…Propagation of CSKs, however, is challenging because CSKs are typically quiescent in adult corneas [ 8 ]. To obtain a large number of CSKs for tissue engineering or cell therapy, the primary cells, after isolation from corneal stromal tissue, are often cultured in the presence of fetal bovine serum (FBS), typically in 5–10% concentration depending on the preferred growth stimulation [ 9 , 10 , 11 , 12 , 13 ]. However, this causes CSKs to become fibroblastic, similar to the in vivo corneal wound healing conditions, in that the surviving CSKs are activated to become stromal fibroblasts (SFs) due to the presence of serum proteins and growth factors that are released after the corneal epithelium and basement membrane are structurally compromised.…”

Advanced surgeries, medicines and materials for corneal regeneration

“…Cells have been not only used to enhance the biomaterial at the time of production but also to secrete ECM, mimicking the formation of corneal stroma during embryogenesis 66 . A characteristic example is the human corneal stromal cell sheet produced by human corneal fibroblasts and subsequently decellularized to manufacture a transparent, thick, cell free and sterile corneal tissue substitute 80,81 .…”

Dimensionality Matters: Exploiting UV-Photopatterned 2D and Two-Photon-Printed 2.5D Contact Guidance Cues to Control Corneal Fibroblast Behavior and Collagen Deposition

“…To this end, contact guidance has been extensively recapitulated and studied using a variety of laboratory setups, for example, using highly defined in vitro substrates presenting grooves (nanoscale [8][9][10][11][12] and microscale [10,11,[13][14][15][16][17][18][19][20][21][22]), lattices [13], micropits [13,22], anisotropic liquid crystalline networks [23], and electrospun fibers [24][25][26][27][28][29]. Depending on the precise dimensions of the (anisotropic) nano-and micro-scale cues, activated keratocytes can spread out and align along the direction of the environmental cue, after which the newly formed collagen is deposited along the same direction [24,30].…”