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Organotypic cell culture system

We developed a novel organotypic long term perfusion cell culture system basing on our cell cultures of dermal fibroblasts and epidermal keratinocytes both isolated from the bovine claw.

The model was developed using commercially available perfusion chambers. Because of some technical limitations we developed and optimised 2 new perfusion chambers allowing long term culture on bio-membranes as basement membrane equivalents. These chambers were developed by Dirk Hoffmann as part of his Dr med vet thesis and assembled by a private engineering group.

Bovine keratinocytes and fibroblasts were obtained from bovine hooves of routinely slaughtered animals from the local abattoir. For the procedure of isolation of these cells see Nebel and Mülling (2002). The cells were cultivated with Dulbecco’s modified Eagle’s medium (Biochrom, Berlin) supplemented with 10% foetal bovine serum (Sigma, Taufkirchen). First, fibroblasts were seeded onto the lower side of the Millicell® insert and allowed to grow for 4 days.

Then the perfusion chamber was mounted by placing the fibroblast into the chamber and subsequently seeding epidermal keratinocytes onto the upper side of the Millicell® insert. The keratinocytes were allowed to adhere for 1 day. Then the cabmber was connected to the tube system with an attached Ismantec 8 cannel pump (Ismatec, Wertheim-Mondfeld) and a gas permeable media bag (Oligene, Berlin). We worked with different flow rates ranging from 0.035ml/min to 0.35ml/min. The system was run for up to 20 days. During this time it was possible to observe the cultivated cells under the light microscope at any time.

The perfusion chamber of Dirk Hoffmann was handled similarly. The only difference was that the keratinocytes were allowed to adhere for 4 days.

In addition, some Millicell® inserts were coated with Matrigel® basement membrane matrix (Becton Dickinson, Heidelberg) using the thin coating method (working instructions of Becton Dickinson) to permit the keratinocytes an improved adherence to the Millicell ® inserts. These Millicell® inserts were handled as described above. Using the Matrigel® thick gel method it was possible to grow cells within a three dimensional matrix. Fibroblasts were dispersed in Matrigel ® subsequently seeded onto the upper side of Millicell® inserts and grown for 4-5 days under standard cell culture conditions. Afterwards, keratinocytes were seeded onto the top of this Matrigel® and allowed to adhere for one day. Then the Millicell® inserts were placed into the perfusion chamber and connected to the fluid flow system.

In our chambers different types of long term culture of fibroblasts and keratinocytes was established and carried out successfully. Keratinocytes showed a tissue specific differentiation and superficial cornification. Morphological examination and protein analysis demonstrated organ specific characteristics. The model is a valid tool for studies of dermo-epidermal cross talk and epidermal proliferation, differentiation and cell death.

Experiments carried out with our culture model revealed the effects of selected cytokines and growth factors on cellular proliferation and differentiation.
With our experimental work we generated initial evidence for the existence of a local paracrine regulatory mechanism in claw tissue potentially involved in the initiation of laminitis. This mechanism explains tissue alterations in damaged tissues as well as the reactive proliferation present in tissues from laminitic cows. IL-1 produced in the epidermis stimulates the production of KGF originating from fibroblasts. KGF is a potent stimulator of keratinocyte proliferation. Proliferation is also stimulated by GM-CSF, suggesting this an other factor in the development of laminitis. TNF-a depresses cellular proliferation and differentiation of keratinocytes, as does IL-1-a. In case of damage of dermal fibroblasts in disease IL- 1 may not cause stimulation but inhibition of keratinocyte proliferation.

Contact

Christoph MUELLING, (assisstant professor)
Tél.: +49-308-3853954
Fax: +49-308-3853480
E-mail