In this article we describe a protocol for growth and expansion of human bronchial epithelial cells from freshly isolated human bronchial segments. This protocol consists of five sections, coding and scratching, a hundred millimeter culture plates, repairing bronchial tissue, explan bronchial tissue transplants, passage of human bronchial epithelial cells growing ciliated, human bronchial epithelial cells on transwells. Before you begin, note that all steps are done in the biological safety cabinet.
Unless otherwise stated, please read the materials, reagents and preparation section and make sure that you have asically prepared. The following coding, stock solution culture, medium Dissociation solutions, Coating and scratching. Hundred millimeter culture plates should be done in the bst.
Keep everything sterile. Place sterile hundred millimeter culture plates and coating solution. In the BST pipette, two mills of coating solution and a hundred millimeter plate swirl around to coat the base of the plate.
Make sure the coating solution covers the base of the plate evenly. Pipette the leftover solution out and use it to coat a second hundred millimeter plate. Repeat for as many plates as needed.
The number of plates needed varies with the number of tissue explants. Make sure the coating solution covers the base of the plates evenly. As Demonstrated.
Cover the plates with Their covers. Allow the plates to dry in the incubator for one to two hours and aspirate access solution before use For bronchial tissue explan. Unused coated culture plates can be placed in a sterile bag and stored at four degrees Celsius for later.
Use spray clean, small, sharp scissors, scalpel and forceps with 70%ethanol and bring into the BSC scratch coated plates by pressing firmly with it scalpel to form deep small XS at least four per plate. If the scratch is not deep enough tissue pieces will not settle in Syringes and will float outside The BSC place tissue in a P three dish. Rinse the isolated human bronchial tissue thoroughly in Bss out.
Open the bronchial pieces. Bring tissue Into the BS and transfer into a sterile hundred millimeter tissue culture plate with called DM EM ham F 12 plus 1%antibiotic anti mycotic. Use the sharp small scissors to cut the tissue Into two to three millimeter cube pieces.
Place one piece of cut tissue in the center of each X with forceps and press. Gently let the tissue pieces or X explants adhere to the plate for a few seconds. And then gently add 10 mils of complete media If the tissue floats After adding media, use forceps to push the tissue into the ridges of the X or make a new X.It may be necessary to make new Xs if the ridges are not deep enough.
Place plates in incubator and change media every three to four days tissues are ready to transplant. When sufficient epithelial cells have grown around tissues to cover one to two centimeter areas, this takes approximately Four weeks. Use a hundred millimeter coated And scratched plates.
Since some tissue explan might not be successful. The number of plates depends on how many pieces of tissue you are transplanting. If you are using plates that have been previously coated and stored at four degrees Celsius, bring to room temperature before use Using forceps carefully pick up tissue from the original plate and place in the center of axis in the new plate and press gently Tissues with no outgrowth should be discarded.
Let tissue adhere to plate. For a few seconds, add media and incubate. Add described earlier for X explan.
So two mils of trips And EDTA stock solution for each hundred millimeter tissue culture plate. Dilute stock with six mils of steryl 0.01 molar PBS and two mils of stock solution to six mils PBS for a final concentration of trypsin at two 50 micrograms per mil and for EDTA at a hundred micrograms Per mil. After moving Explants to a new plate, aspirate media from plates with one to two centimeter Rings of cell.
Add eight mils of warm diluted drips and edt a solution to each a hundred millimeter plate. And place an incubator at 37 degrees Celsius for three to 15 minutes. Check frequently.
Leaving trypsin on too long will damage the cells. Check plates under a microscope to be sure most of the cells have been lifted, cells will round Up and detach as shown at eight Mils of warmth. 10%FBS in media per plate to inactivate thein EDT a solution.
Combine volumes of all plates into one tube or separate tubes of different tissues. Count the cells using a Hema Cytometer centrifuge at a hundred G for five minutes. A cell pellet will form at the base of the tube resuspend cell pellet in complete media to the desired cell concentration.
Place each two to 3 million cells per T 75 flask and add complete media as required. Incubate and change media. Every three to four weeks, cells grow to sub confluence in D 75 flasks.
In about four weeks, the cells should be lifted and expanded or used at sub confluence to prevent sene. Start with preparing the permeable membranes of the transwells. Use the 6.5 millimeter inserts that fit into the 24 well multiple well plates.
Add medium to both sides of the membrane. Point six mils on the bottom and 0.1 mils on the top. Incubate one hour in cell culture incubator.
Carefully Remove the medium from both sides of the membrane. Starting with the basal volume first carefully means the membrane can be easily damaged. So pipette the medium down the side of the culture insert, use the volume recommended.
Not doing so will result in leaking over Of media to other well to seed cells. After pre incubation of permeable support, pipette carefully your cell suspension into the epical side of the membrane for 6.5 millimeter wells at 0.1 mils of cell suspension to the epical side of the membrane. Pipette 0.6 M, medium on the basal side of the membrane.
Feed the cells from the epical and basal sides for 10 days. To establish a well differentiated culture exchange, medium twice a week. Using the demonstrated method, remove basal volume.
First, replace epical volume, add 0.6 mils of medium to the basal side of the membrane. This method promotes cell attachment to membrane and prevents cells from being exposed to air for long periods of time. Exchange the media quickly and put back an incubator.
Create an air liquid interface on day 10 by removing the AAL medium and replacing the 0.6 mil of medium on the basal side of the membrane. Maintain cells in air liquid interface for six weeks change media twice a week. Ciliated cells will start appearing four weeks after creation of the ear.
Liquid interface. Cells will achieve uniform differentiation into ciliated cells. Six weeks after creation of The ear liquid interface.
Human bronchial Segments with healthy epithelial cells from explan and transplants. In our study, successful cultures from explan of the bronchial tissue occurred in eight out of nine tissues. As one of the explan cultures got infected, the rings of epithelial cells reached one to two centimeter radius in three to four weeks.
Successful X explan were cultured again up to six times. All successful cultures show evidence of cell migration within 48 hours of starting the transplants, or X explan as shown in B cells have a cobblestone appearance that is distinct for these epithelial cells as shown in B.And in subsequent figures, immuno staining of cytosine preparations of cells from explan and cells seeded on cover slips revealed that all cells were positive for the epithelium specific cytokeratin, human subculture bronchial cells grown to sub confluence and T 75 flasks are shown here. Note the cobblestone appearance.
Human subculture bronchial cells showed uniform positive immunostaining for kerin and E.Can him immunostaining with specific antibodies against other cell types did not show any indication of contamination of cultures by fibroblasts, mesenchymal, or endothelial cells. For more details, please refer to figure five and its legend in the text cells cultured on permeable polyester membranes or transwells with or liquid interface differentiated into ciliated epithelium as demonstrated here, discard cells and theirs or transplants if cells with a different morphology as demonstrated Here start Appearing. In this protocol, We presented detailed methods for culture and expansion of primary human bronchial epithelial cells.
Human cell-based studies are needed for determining important pathways of human lung disease mechanisms and for toxicity and pharmacologic screening of novel drug. We hope the methods described here will encourage you to culture human bronchial epithelial cells from explants and to use these cells in your study systems. Thank you and good luck.
