Скачать или смотреть Millicell® Hanging Inserts – How to use – Chapter 3: Organotypic Air-Liquid Interface 3D Skin

As researchers transition to animal-free testing, in vitro human cell culture models are a sustainable, effective, and reliable solution. With the Millicell® Cell Culture Inserts, you can create biologically relevant skin models. In this video, we will show you how to generate the organotypic air-liquid interface, or ALI, 3D skin cultures. This serves as an in vitro model of the human epidermis and can be used in applications for cosmetic testing to clinical screening.

Here we present a diagram of the steps to generate an ALI skin model. In the first step of generating this model, we will create stratification of the skin. Mix collagen and Fibroblast cells and place on top of the membrane.

Step One – Prepare Collagen Bed with Fibrolasts
Add collagen to empty tube
Add 5x reconstruction buffer to mixture
Add fibroblasts to mixture and gently stir

Collagen is very sticky and can solidify quickly, the best method is to add the mixture to the middle of the insert while ensuring the tip does not touch the membrane. This ensures an even collagen bed. Polymerize the collagen bed for 30 minutes in the tissue culture incubator. After solidifying the collagen, add in the primary cells. Now, place the plate into an incubator.

Step Two – Add Keratinocytes on top of Collagen & Fibroblast Bed

After the polyermization, add the keratinocytes into apical side.

Step Three – Add the Media to the Basolateral Side

Submerge the culture for two additional days in growth media. More details on media exchange and respective volumes can be found at http://ms.spr.ly/6055sa4HB

Step Four – Air Lift and Add Differentiation Medium

On day 4 we will change the media to the 3dGRO® differentiation medium. Remove the apical media. It is important that you do not add any liquid to expose cells to the air. Then, exchange the basolateral media into the 3dGRO® medium. The volume will depend on your plate height. Ensure the media in the bottom, or basolateral side, is lower than the collagen bed.

Tip: Change medium every day for ten days

Step Five – Fix Cells

Now, to prepare to section and stain the membranes, fix with a standard fixative solution for tissue processing.

Tip: Avoid using acetone to fix cells, as it may not be compatible with plastic cultureware

We will be using 4% paraformaldehyde for 15 minutes.

Step Six – Process for Sectioning and H&E Staining

Flip the insert upside down and use a #11 blade to excise the membrane. Gently hold the membrane with forceps, noting the side of the membrane with cells. For sectioning such as H&E Staining, place the membrane in a microfuge tube for further post processing.

In following these steps, you can expect to see three separate layers in your culture. Ensuring confidence when performing downstream assays. You can also stain for Ki67 and Filaggrin to confirm the proliferation and differentiation of the skin model. Downstream assays can be performed on the skin model such as permeability assays, TEER readings, drug screens, and wound healing.

For more information, please contact your account manager or visit http://ms.spr.ly/6056sa4H8