Traditional diffusal culture methods often fail to recapitulate the complex environment of living organs, leading to the loss of tissue-specific markers and functions. We have developed an open-top chip that combines diffusal culture and microfluidic principle to mimic the microenvironment of epithelial tissues closely. By including biochemical and biomechanical cues that are naturally present in living organs but often neglected by traditional in vitro models, the open-top chip represent a better alternative between closed systems.
Demonstrating the procedure will be Adya Panchal today, a research student in my laboratory. To begin, bring the required reagents under the biosafety cabinet on ice. Culture tissue-specific mesenchymal cells to obtain 80%to 90%confluency and then dissociate the cells using trypsin or other methods as per the manufacturer's recommendations.
Pellet the cells at 250g for five minutes at 24 degrees Celsius. Resuspend the cell pellet in 225 microliters each of ice-cold 10X EMEM and reconstruction buffer. Mix the cells by pipetting and add 1, 800 microliters of ice-cold Collagen 1 solution.
Next, mix the pre-gel solution on ice by pipetting up and down five to six times. Neutralize the pre-gel solution with 18 microliters of one normal sodium hydroxide, mix gently by pipetting, and then pipette 150 microliters of cell-laden hydrogel into the central chamber of the open-top chip while avoiding bubbles. Group the chips into separate Petri dishes, including a centrifuge tube cap filled with two milliliters of sterile double-distilled water in each Petri dish, and incubate the Petri dishes at 37 degrees Celsius and 5%carbon dioxide.
To perform surface micropatterning of the stromal hydrogel, pipette 20 microliters of the neutralized Collagen 1 pre-gel solution on the patterned surface of a sterile 3D-printed stamp and insert the stamp inside of the open-top chamber while the stromal hydrogel is still in a liquid form. Remove any hydrogel residue that may spill from the top of the open-top chamber using an aspirator. Group all chips into separate Petri dishes with a conical tube cap filled with water as demonstrated earlier.
And incubate all Petri dishes at 37 degrees Celsius and 5%carbon dioxide for 90 minutes. At the end of the incubation, bring the chips back under the biosafety cabinet and gently remove the stamps using precision tweezers to reduce the risk of damaging the hydrogel. Once the cultured cells attain 80%to 90%confluency, dissociate the cells using proteolytic enzyme procedures as recommended by the cell provider.
After dissociation, pellet the cells by centrifugation and resuspend the epithelial cells to the appropriate cell fragment density as described in the manuscript. To seed the epithelial cell, transfer the chips from the incubator into the biosafety cabinet and rinse the stromal surface three times with 100 microliters of fresh epithelial cell culture medium to remove any excess of coating solution. Once the rinsing medium is aspirated, seed the hydrogel surface with 50 microliters of the epithelial cell suspension using appropriate cell density, and then transfer the chips back into the incubator for two hours.
To remove cellular debris, gently rinse the hydrogel surface with the cell culture medium twice, refresh the medium by autoclaving in sealed autoclavable containers, and connect the chips to the peristaltic pump. Pause the peristaltic pump, carefully disconnect the chips from the pump, and extract the chip housing carrier. After transferring the chips from the incubator to the biosafety cabinet, remove the volume of the medium left into the top reservoir.
Then, aspirate all the medium from the top microfluidic channel gently and clamp the short microfluidic tubing connected to the top inlets using binder clips to reduce media evaporation and maintenance of air-liquid interface, or ALI. Place the open-top chip on the housing carrier back into the incubator. Reconnect the chips to the peristaltic pump and resume the flow by starting the peristaltic pump.
Rinse the vascular chamber with endothelial cell culture medium and then seed 25 microliters of endothelial cell suspension. Flip the chips upside down to allow endothelial cells to attach to the upper surface of the microfluidic chamber. Group the chips into Petri dishes.
Place them back into the incubator as demonstrated earlier and let the endothelial cells attach for one hour. At the end of the incubation, transfer the chips from the incubator to the biosafety cabinet and rinse the vascular channel with endothelial cell culture medium twice to remove cellular debris. Lay the chips flat to facilitate the adhesion of the endothelial cells to the bottom surface of the vascular channel.
Fill the vascular medium reservoir with the degassed vascular cell culture medium under the biosafety cabinet. Place the chips back inside the chip housing carrier and reconnect the chips to the medium reservoirs on one end and to the peristaltic pump on the other. Inspect the microfluidic connections to ensure all the chips are correctly connected and no visible droplets of medium dripping.
Then, resume fluid flow by starting the peristaltic pump. The micropatterned gel surface with and without cells is visualized. Histological analysis revealed mature multilayered stratified epidermis differentiated on chip.
The top view picture of the skin chip showed the presence of the fibroblasts inside the dermal layer. PECAM-1, VE-cadherin, and von Willebrand showed the differentiation of human microvascular endothelial cells co-cultured in the open-top skin chip. MUC5AC, alpha and beta tubulin, chloro cell protein 16, p63, and ZO-1 fluorescent staining showed mature airway epithelium.
The beating Cilia and differentiated mature pseudo-stratified epithelium were observed on open-top airway chip. Type I, Type II, and E-cadherin fluorescent staining showed the presence of mature pneumocytes on chip. Electron microscopy revealed the presence of microvilli and lysosomal vesicles, evidence of mature alveolar phenotype.
The histological analysis showed the presence of flat squamous cells consistent with the Type I phenotype, and cuboidal cobblestone-like cells coherent with the Type II phenotype, and fibroblasts inside the dermal layer. The presence of enterocytes and the mature goblet was confirmed by mucin 2 and E-cadherin staining. The fibroblasts inside the dermal layer confirmed the presence of a simple columnar epithelium.
All reagents must be cold and the surfaces of the gel should be adequately rinsed to obtain an even surface. It's important to remove any non adhering cells and debris to obtain even cell adhesion and a compact foul monolayer and vascular wall. A similar approach can be employed to generate other types of epithelial organ models, such as the intestine and the lung, to assess how specific drugs or environmental factors may affect the homeostasis of human tissues, with a particular emphasis on epithelial and vascular barrier function.
With easy access to the efficacy phase of the stroma compartment, researching can now generate specific face patterns and reproduce functional tissue architecture, such as the cryptviveoli, which is difficult to achieve with other devices.
