Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

Transcript

The overall goal of this procedure is to enhance extracellular matrix deposition and improve on current 2D and 3D skin models using macromolecular crowding. Crowding improves the generation of cell-derived matrices, and mature organotypic cultures in a condensed time frame. This method can answer key questions in the field of tissue engineering because it offers improved ways of generating cell-derived extracellular matrices for the bioengineering of skin and other tissues.

The main advantage of this technique is that cell-derived matrices and organotypic skin constructs can be generated in a significantly condensed time frame when applying macromolecular crowding to these cultures. The technique of interference reflection microscopy allows you to visualize the total extracellular matrix deposition without the need of antibody staining. Applications of these techniques will extend from studies of extracellular matrix deposition by skin cells to improved wound therapies, especially with the generation of better skin equivalents and tissue culture for grafting.

To begin, seed 50, 000 primary fibroblasts, primary keratinocytes, or a co-culture of both, in one milliliter of medium pro well, into the wells of a 24-well plate. Allow the cells to adhere in a 37 degree Celsius, 5%carbon dioxide incubator overnight. Then, discard the old medium and replace it with one milliliter of fresh medium containing macromolecular crowders and 100-micromolar ascorbic acid.

Incubate the cultures for six days, changing the medium every two days. Carry out immunofluorescence and Western blotting according to the text protocol. To make and use a cell-derived matrix, after culturing the cells for six days with molecular crowders and ascorbic acid as just demonstrated, decellularize the cell layers to obtain a cell-derived matrix by first using 1X PBS to wash the cell layers twice.

Aspirate the PBS, and then add 250 microliter of 0.5%sodium deoxycholate and incubate on ice for 10 minutes. Aspirate the cell lysate, then add an additional 250 microliter of 0.5%sodium deoxycholate and repeat the 10-minute incubation and aspiration two more times. Use distilled water to wash the cell-derived matrix twice.

Aspirate the water, and then add 1X PBS. Store the matrix at four degrees Celsius for up to one week. To prepare a secondary cell suspension, aspirate the PBS from the matrix, and seed 50, 000 keratinocytes on top of the F mat for example.

Allow the cells to adhere overnight. To carry out interference reflection microscopy, or RIM, on a cell-derived matrix, perform image acquisition using a con-focal microscope according to the text protocol. To cast a collagen gel with fibroblasts encapsulated in a cell culture insert, aliquot 10 milliliters of rat tail collagen type one into a cold beaker.

Add one milliliter of cold DMEM and 0.5 milliliters of one molar sodium hydroxide to neutralize the acidic collagen. Then add 0.5 milliliters of fibroblast suspension. Using a cold pipette, evenly divide the 12 milliliters of solution into cell culture inserts in a six-well culture plate.

Incubate the gel at 37 degrees Celsius and 5%carbon dioxide for one hour. Once the gel has solidified, submerge it in fibroblast medium or FM.Then after incubating it for 24 hours, aspirate the FM.Use four milliliters of fresh medium containing macromolecular crowders to replace the old FM by adding two milliliters to the interior of the cell culture insert and two milliliters to the outside of the cell culture insert. Following a 24-hour incubation, use 0.125%trypsin cheating agent to trysonize the keratinocytes.

Tap the sides of the flask gently to dislodge the cells and add serum-containing medium to neutralize the trypsin. After counting the cells, prepare a keratinocyte suspension. Next, aspirate the medium from the cell culture inserts, and add the keratinocytes to the top of the gel.

Incubate the gel for one hour. After the keratinocytes have attached to the gel surface, use two milliliters of KSFM, or CTN-57 serum-free medium to submerge the cell inside the gel culture insert and add two milliliters of FM to the outside of the cell culture insert. Following a 24-hour incubation, discard the old medium and replace it with fresh medium containing macromolecular crowders.

After seven days in the submerged culture, to raise the culture to an air-liquid interface, transfer the culture insert to a deep-well plate. Add 10 milliliters of stratification medium to the outside of the cell culture insert, keeping the interior of the insert dry. Finally, incubate the cultures and change the medium every three days for 14 days.

Harvest and process them according to the text protocol. As shown here, macromolecular crowding allowed fibroblasts to deposit more extracellular matrix or ECM as compared to control cultures. Upon secularization, it was evident that fibroblasts were the main depositors of collagen one, collagen four, and fibronectin.

This figure demonstrates that is was fibroblasts rather than keratinocytes that were the main depositors of collagen seven, and this is the first report of successful collagen seven deposition in vitro. Using RIM, the full extent of the cell-derived matrix was captured. An overlay of the RIM image with antibody staining shows the relative quantity of that ECM component in relation to the total amount of ECM.

In a 3D in vitro skin model, macromolecular crowding or MMC, shortened the culture time from five weeks to three, to obtain a mature organotypic skin co-culture. As seen here, by H and E staining, at three weeks, the culture with MMC consisted of a pleuristratified epidermis and a stromol ridge dermis. In addition, intense and continuous collagen seven immnunostaining was detected at the dermalepidermal junction of crowded cell cultures as compared to a weak and spotty collagen seven immunostaining in control cultures.

Transmission electron microscopy also show the presence of anchoring fibrils in organotypic cultures generated with MMC, showing functional collagen seven. Cell-derived matrices could be used for secondary cell seeding purposes as an alternative scaffold as the complexity of the extracellular matrix is maintained, and it is produced by cells themselves. Organotypic skin cultures, which are generated under macromolecular crowding, could be used as an improved 3D skin model as it contain a stromal ridge dermis, a major dermalepidermal junction, and a pleuristratifed epidermis.

Organotypic, or skin equivalent cultures generated with macromolecular crowders can also be used for skin grafting because they have a better developed dermalepidermal junction, which is likely to improve graft success. In addition to enhanced extracellular matrix deposition, macromolecular crowding can be applied to other in vitro systems to enhance reactions with are rate limited, without having to increase the amount of reagents. For example, polymerase chain reactions.

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Journal (Bioengineering)

Journal (Bioengineering)

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

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