Human Organ Culture: Updating the Approach to Bridge the Gap from In Vitro to In Vivo in Inflammation, Cancer, and Stem Cell Biology

Abstract

Human studies, critical for developing new diagnostics and therapeutics, are limited by ethical and logistical issues, and preclinical animal studies are often poor predictors of human responses. Standard human cell cultures can address some of these concerns but the absence of the normal tissue microenvironment can alter cellular responses. Three-dimensional cultures that position cells on synthetic matrices, or organoid or organ-on-a-chip cultures, in which different cell spontaneously organize contacts with other cells and natural matrix only partly overcome this limitation. Here, we review how human organ cultures (HOCs) can more faithfully preserve in vivo tissue architecture and can better represent disease-associated changes. We will specifically describe how HOCs can be combined with both traditional and more modern morphological techniques to reveal how anatomic location can alter cellular responses at a molecular level and permit comparisons among different cells and different cell types within the same tissue. Examples are provided involving use of HOCs to study inflammation, cancer, and stem cell biology.

Keywords: cancer; human; inflammation; organ culture; stem cells.

Figure 1

Schematic diagram of human organ culture (HOC) setup. Tissue is immersed in a sterile universal container with tissue culture medium (M199 supplemented with 5% heat-inactivated fetal calf serum, 100 U/ml penicillin/streptomycin, 2.2 mM l-glutamine) (A). Tissue is rinsed in sterile phosphate-buffered saline (PBS) to remove excess blood (B). Tissue is dissected under a dissecting microscope using two sterilized carbon steel razor blades single-edged stuck together creating a gap of <1 mm³ (C). Tissue fragments are immersed in sterile 96-well plate containing 200 µl medium and sterile culture insert, treated as indicated, or left untreated and maintained in a 37°C incubator (95% air, 5% CO₂) for the indicated time periods (usually less than 24 h) (D). Upon harvest, HOCs are either immersed in fixative (paraformaldehyde for light microscopy or gluteradehyde for electron microscopy) or in RNA later or snap-frozen in isopentane-cooled in liquid nitrogen for molecular biological/biochemical analysis (E). IHC, immunohistochemistry; IF, immunofluorescence; PLA, in situ proximity ligation assay; TUNEL, terminal transferase-mediated d-UTP nick end labeling; TEM, transmission eIectron microscopy; IGEM, immunogold EM.

Figure 2

(A) RCC organ cultures treated with recombinant human TNF for 3 h and subjected to immunogold electron microscopy. Colloidal gold particles representing localization of phospho-MLKL (Ser358) (5 nm) and phospho-dyamin-related protein-1 (Drp1) (Ser616) (10 nm) and are detected in mitochondria (m). Mag: 105k×. (B) Representative light micrograph of RCC organ cultures treated with recombinant human TNF for 3 h were subjected to in situ hybridization using anti-sense probe to RIPK3 conjugated with Digoxigenin (DIG). Intense blue staining using anti-DIG-alkaline phosphatase and BCIP/NBT substrate (blue color) is seen in sites of gene expression within the cytoplasm of tumor cells. Original mag: 63×. (C) Representative confocal image of RCC organ cultures treated with recombinant human TNF for 3 h and subjected to in situ proximity assay to determine interaction between RIPK3 and phosphorylated-MLKL (Ser358). Numerous strong red fluorescent spots are evident within the cytoplasm of malignant TECs (mTECs) indicating close proximity of RIP3/pMLKL (<40 nm). Original mag: 63×. TNF, tumor necrosis factor.