Histotypic 組織培養

Histotypic tissue culture allows cells to be grown in three dimensions, thereby creating in-vitro tissue morphologies that closely mimic realistic tissue function, which can be used as viable constructs for tissue repair. These cultures are typically three dimensional structures consisting of a single cell type grown in high density. The three dimensional structure, also known as the scaffold, mimics the natural extracellular matrix. Depending on the cell type used, scaffolds can be designed for a specific application and typically act as a template for bio-mimetic tissue. This video will introduce the fundamentals of histotypic tissue cultures, a procedure for the isolation of cells, and the fabrication and cellularization of a porous silk tissue scaffold to mimic cardiac tissue.

All tissues consist of two fundamental components, the extracellular matrix and the tissue-specific cells that inhabit it. The extracellular matrix is a network of structural proteins that create a three dimensional environment for cells to occupy, and the cells within it are meant to recapitulate the native physiological processes of the tissue. Currently, a common technique utilized to model tissue is two dimensional tissue culture, where cells are dispensed onto a flat substrate and allowed to form a thin film. In general, this method is not reliable for maintaining an in vivo phenotype, organ-specific functions, and cell to cell or cell to substrate contextual interactions. Histotypic tissue culture alleviates those limitations by providing a 3D scaffold for cells to grow on, resulting in a dense network of cells that more closely mimics native cell morphologies and facilitates the development of realistic intercellular networks and communication pathways. A variety of 3D polymer networks, including hydrogels and electrospun silk mats, offer convenient ways to culture tissue-specific cells in three dimensions. In order to populate these scaffolds, cells must be isolated. Primary cells used in this video are harvested from live tissue, which is minced and then digested in an enzyme solution to separate the target cells from the extracellular matrix. Once the cells are isolated, there are two techniques used to seed the scaffolds. The droplet technique involves pipetting a solution of cells onto the scaffold at a slow and constant rate. The second, or cell suspension technique, submerges the scaffold in a cell suspension. Often, the scaffold and suspension are shaken to encourage cell migration into the matrix. Both techniques result in bio-engineered constructs with high cell densities. The following procedures will involve the isolation of cardiac cells and the cell suspension technique to create a cardiac cell-specific scaffold, as it will retain the native heart tissue morphology.

To begin the process of isolating cells from donor tissue, start by ensuring that the work area and dissection instruments are sterilized. Then, place a sterile drape on the work surface in the bio-safety cabinet. Place the sterile surgical instruments onto the drape without touching them, and then open a sterile number 20 scalpel blade. After euthanizing the specimen, sterilize the surgical area with a betadine-soaked gauze pad. When ready, secure the sample and begin the surgical procedure to isolate the tissue of interest. In this case, it will be the heart. Once excised, place the tissue on ice in the Petri dish containing PBS glucose. Remove any residual blood or connective tissue, and then transfer the tissue to a Petri dish with fresh PBS glucose. Then, using sterile micro-scissors and forceps, carefully mince the tissue samples into roughly 1 cubic millimeter pieces. Using a pipette, transfer the pieces and buffer to a conical tube. Then, remove all but 10 milliliters of buffer. Add 7 milliliters of collagenase solution, and then shake the mixture at 37 degrees Celsius for 7 minutes. Then, gently pipette 10 times to break up the tissue pieces. Allow the pieces to settle, and then aspirate the liquid and discard it. Next, repeat the digestion and gently pipette the solution to break up the tissue pieces. After the pieces have settled, draw off the supernatant and collect it in a separate 50 milliliter conical tube. Then, add 10 milliliters of STOP solution to each conical tube containing supernatant to stop the digestion.

Now that the primary cells have been isolated, let's fabricate a porous silk tissue scaffold. To begin, pour 30 milliliters of the silk solution into a mold. Next, scatter 60 grams of sieved sodium chloride evenly over the solution. Then, allow the silk to polymerize undisturbed at room temperature for 48 hours. Then, place the mold in a 60 degree oven for 1 hour to finalize cross-linking and evaporate any remaining liquid. Once polymerized, immerse the mold in a beaker of distilled water for 48 hours to leech out the salt. Then, remove the scaffold from the mold and cut small discs with a 5 millimeter biopsy punch. Trim the discs to a height of 2 millimeters, and finally, remove the centers of each piece with a 2 millimeter biopsy punch to create a ring. Lastly, autoclave the scaffolds in a wet cycle for 20 minutes.

With the scaffold prepared, let's begin the cell seeding process. First, place one sterile scaffold per well in a 96 well plate. Then, add cell culture medium to immerse the scaffolds and incubate at 37 degrees Celsius in a tissue culture incubator to equilibrate them for at least 30 minutes. Following incubation, aspirate the excess medium and then add the isolated primary cell suspension to the scaffolds. Next, return the plate to the incubator and leave overnight for the cells to attach to the scaffolds. On the following morning, carefully aspirate the non-attached cells and replace with 200 microliters of fresh cell culture medium per well. The resulting scaffold is a porous construct with a high cell density ready to be used.

Now that you have learned how to perform histotypic tissue culture, let's look at some practical applications of these materials. Histotypic tissue culture can create cellular micro-environments that mimic native tissues, and as a result are able to provide a suitable model for the study of cellular behavior concerning a single cell type. For example, 3D fiber in scaffolds, which more accurately mimic the stem cell niche found in vivo, can be seeded with pluripotent stem cells to screen for biological cues and determine their effects on stem cell differentiation. This work may ultimately provide a greater understanding of how stem cells differentiate and may offer insights into enhancing cell differentiation and regeneration for tissue engineering applications. Like dynamic cultures, mechanical conditioning can also enhance the 3D tissue scaffold by subjecting it to various mechanical loads that natural tissue may experience in vivo. By applying compression and biaxial loads during cell growth, the cell morphology and extracellular matrix is altered to reflect those mechanical loads. This results in a preconditioned bio-engineered tissue scaffold with a cellular structure resembling native tissue, making it ideal for implantation in areas that may experience similar mechanical forces. Finally, engineered tissue constructs may also be used to replace or repair tissue defects. In order to achieve this, the tissue scaffold must be first vascularized, thereby allowing blood to freely move through the construct. Once vascularized, the scaffold can be transferred and implanted into the tissue defect to initiate repair. Successful grafting can later be confirmed through histology, which reveals whether or not the tissue construct completely repaired the damaged area.

You've just watched Jove's introduction to histotypic tissue culture. You should now understand how simple 3D structures are prepared, how primary cells are isolated and seeded onto a scaffold, and the various applications of these cultures in the bio-engineering field. Thanks for watching.