Stem and progenitor cells isolated from human limbal tissue can be cultured in vitro as spheres. These spheres have potential for use as transplantable elements for the repopulation of corneal tissue (Mathan et al., 2016). Herein we describe the detailed protocol for the implantation of human corneal spheres into cadaveric human corneal tissue. This protocol describes the procedure for sphere formation and culture, preparation of tissue for sphere implantation, corneal limbus microsurgery and sphere implantation.

Fluorescence live-cell imaging by single molecule localization microscopy (SMLM) or fluorescence lifetime imaging microscopy (FLIM) in principle allows for the spatio-temporal observation of molecular patterns in individual, living cells. However, the dynamics of molecules within cells hamper their precise observation. We present here a detailed protocol for consecutive cycles of reversible cryo-arrest of living cells on a microscope that allows for a precise determination of the evolution of molecular patterns within individual living cells. The usefulness of this approach has been demonstrated by observing ligand-induced clustering of receptor tyrosine kinases as well as their activity patterns by SMLM and FLIM (Masip et al., 2016).

Adipose-derived stromal/stem cells (ASCs) are multipotent cells that can be isolated from adipose tissue. Studies have shown that cells have the capacity to self-renew and differentiate into adipocyte, chondrocyte, myocyte, and osteoblast lineages. Thus, significant interest regarding their use for regenerative purposes to restore aging or damaged tissue has grown in recent decades. These cells have also been shown to immunomodulate the microenvironment and secrete abundant growth factors, which minimize inflammation and aid repair and regeneration. ASCs can be readily isolated from the stromal vascular fraction (SVF) of lipoaspirates. Given their ease of accessibility, bountiful source, and potential application in regenerative medicine and tissue engineering, there is growing interest in the characterization and utilization of ASCs. This protocol describes the isolation of ASCs from adult human adipose tissue as well as methods for culture maintenance including expansion and cryopreservation.

Hydrogels are an ideal medium for the expansion of cells in three dimensions. The ability to induce cell expansion and differentiation in a controlled manner is a key goal in tissue engineering. Here we describe a detailed method for producing hydrogels from soft tissues with an emphasis on adipose tissue. In this method, soluble, extractable proteins are recovered from the tissue and stored while the remaining insoluble tissue is processed and solubilised. Once the tissue has been sufficiently solubilised, the extracted proteins are added. The resulting product is a thermosensitive hydrogel with proteins representative of the native tissue. This method addresses common issues encountered when working with some biomaterials, such as high lipid content, DNA contamination, and finding an appropriate sterilisation method. Although the focus of this article is on adipose tissue, using this method we have produced hydrogels from other soft tissues including muscle, liver, and cardiac tissue.